Neprilysin inhibitors

ABSTRACT

In one aspect, the invention relates to compounds having the formula: 
                         
where X, R a , R b , R 2 , and R 7  are as defined in the specification, or a pharmaceutically acceptable salt thereof. These compounds are prodrugs of compounds having neprilysin inhibition activity. In another aspect, the invention relates to pharmaceutical compositions comprising these compounds; methods of using these compounds; and processes and intermediates for preparing these compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/497,508, filed on Apr. 26, 2017, now allowed; which is a continuationof U.S. application Ser. No. 14/812,095, filed on Jul. 29, 2015, nowU.S. Pat. No. 9,670,186; which is a continuation of U.S. applicationSer. No. 13/961,269, filed on Aug. 7, 2013, now U.S. Pat. No. 9,126,956;which application claims the benefit of U.S. Provisional Application No.61/680,804, filed on Aug. 8, 2012 and U.S. Provisional Application No.61/774,163, filed on Mar. 7, 2013; the entire disclosures of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to novel compounds havingneprilysin-inhibition activity or which are metabolized in vivo tocompounds having such activity. The invention also relates topharmaceutical compositions comprising these compounds, processes andintermediates for preparing these compounds and methods of using thesecompounds to treat diseases such as hypertension, heart failure,pulmonary hypertension, and renal disease.

State of the Art

Commonly-assigned U.S. Patent Publication No. 2012/0213806, filed onFeb. 16, 2012 to Fleury et al., describes novel compounds that haveactivity as neprilysin inhibitors, the disclosure of which isincorporated herein by reference. In particular, compounds of the genus:

are described. Depending upon the variables, compounds within this genuscan be referred to as being in the active form or as being a prodrug,which is metabolized in vivo to generate the active form of thecompound.

In spite of these compounds however, there remains a need for compoundsand prodrugs within this genus that have different metabolic andcleavage properties. For example, there remains a need for activecompounds and/or prodrug compounds having improved oral absorption andfor prodrug compounds that undergo rapid cleavage to form the activecompound. This invention is directed to that need.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a compound of formula I:

where:

(i) X is

and

-   -   (a) R^(a) and R^(b) are H; R² is H; and R⁷ is selected from        —CH₂CF₂CH₃, —CH₂CF₂CF₃, —(CH₂)₅CH₃, —(CH₂)₆CH₃, and

or R² is —C₁₋₆alkyl or —C(O)—C₁₋₆alkyl, and R⁷ is H; or

-   -   (b) R^(a) is selected from —CH₃, —OCH₃, and Cl and R^(b) is H;        or R^(a) is selected from H, —CH₃, Cl, and F, and R^(b) is Cl;        or R^(a) is H and R^(b) is selected from —CH₃ and —CN; R² is        selected from H, —C₁₋₆alkyl, —(CH₂)₂₋₃OR^(e), and        —(CH₂)₂₋₃NR^(e)R^(e); and R⁷ is selected from H, —C₁₋₆alkyl,        —[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl, —CH₂OC(O)CHR^(d)—NH₂,        —CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, —CHR^(c)OC(O)O—C₂₋₄alkyl,        —CHR^(c)OC(O)O-cyclohexyl, —CH₂CH(NH₂)C(O)OCH₃,        —C₂₋₄alkylene-N(CH₃)₂, —C₀₋₆alkylenemorpholinyl, and

or

-   -   (c) R^(a) is H and R^(b) is F; or R^(a) is F and R^(b) is H; R²        is selected from H, —C₁₋₆alkyl, —(CH₂)₂₋₃OR^(e), and        —(CH₂)₂₋₃NR^(e)R^(e); and R⁷ is selected from —C₁₋₆alkyl,        —[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl, —CH₂OC(O)CHR^(d)—NH₂,        —CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, —CHR^(c)OC(O)O—C₂₋₄alkyl,        —CHR^(c)OC(O)O-cyclohexyl, —CH₂CH(NH₂)C(O)OCH₃,        —C₂₋₄alkylene-N(CH₃)₂, —C₀₋₆alkylenemorpholinyl, and

or

(ii) X is

and

-   -   (a) R^(a) is Cl and R^(b) is H; or R^(a) is H and R^(b) is        selected from Cl, F, —CH₃, and —CN; or R^(a) is F and R^(b) is        Cl; R² is selected from H, —C₁₋₆alkyl, —(CH₂)₂₋₃OR^(e), and        —(CH₂)₂₋₃NR^(e)R^(e); R⁴ is selected from —OH, —OCH₃, —OCH₂CH₃,        and —C₁₋₄alkyl; and R⁷ is selected from H, —C₁₋₆alkyl,        —[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl, —CH₂OC(O)CHR^(d)—NH₂,        —CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, —CHR^(c)OC(O)O—C₂₋₄alkyl,        —CHR^(c)OC(O)O-cyclohexyl, —CH₂CH(NH₂)C(O)OCH₃,        —C₂₋₄alkylene-N(CH₃)₂, —C₀₋₆alkylenemorpholinyl, and

or

-   -   (b) R^(a) is F and R^(b) is H; R² is H; R⁴ is —OH; and R⁷ is        selected from —C₁₋₆alkyl, —[(CH₂)₂O]₁₋₃CH₃,        —CHR^(c)OC(O)—C₁₋₄alkyl, —CH₂OC(O)CHR^(d)—NH₂,        —CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, —CHR^(c)OC(O)O—C₂₋₄alkyl,        —CHR^(c)OC(O)O-cyclohexyl, —CH₂CH(NH₂)C(O)OCH₃,        —C₂₋₄alkylene-N(CH₃)₂, —C₀₋₆alkylenemorpholinyl, and

or

(iii) X is

and

-   -   (a) R^(a) is Cl and R^(b) is H; or R^(a) is H and R^(b) is        selected from Cl, F, —CH₃, and —CN; or R^(a) is F and R^(b) is        Cl; R² is selected from H, —C₁₋₆alkyl, —(CH₂)₂₋₃OR^(e), and        —(CH₂)₂₋₃NR^(e)R^(e); and R⁷ is selected from H, —C₁₋₆alkyl,        —[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl, —CH₂OC(O)CHR^(d)—NH₂,        —CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, —CHR^(c)OC(O)O—C₂₋₄alkyl,        —CHR^(c)OC(O)O-cyclohexyl, —CH₂CH(NH₂)C(O)OCH₃,        —C₂₋₄alkylene-N(CH₃)₂, —C₀₋₆alkylenemorpholinyl, and

or

-   -   (b) R^(a) is F and R^(b) is H; R² is H; and R⁷ is selected from        —C₁₋₆alkyl, —[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,        —CH₂OC(O)CHR^(d)—NH₂, —CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl,        —CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,        —CH₂CH(NH₂)C(O)OCH₃, —C₂₋₄alkylene-N(CH₃)₂,        —C₀₋₆alkylenemorpholinyl, and

or

(iv) X is

-   -   (a) R^(a) and R^(b) are H; R² is selected from —C₁₋₆alkyl,        —(CH₂)₂₋₃OR^(e), and —(CH₂)₂₋₃NR^(e)R^(e); R³ is selected from        —OH, —OCH₃, —OCH₂CH₃, and —C₁₋₄alkyl; and R⁷ is H; or    -   (b) R^(a) is selected from Cl and F and R^(b) is H; or R^(a) is        H and R^(b) is selected from Cl, F, —CH₃, and —CN; or R^(a) is F        and R^(b) is Cl; R² is selected from H, —C₁₋₆alkyl,        —(CH₂)₂₋₃OR^(e), and —(CH₂)₂₋₃NR^(e)R^(e); R³ is selected from        —OH, —OCH₃, —OCH₂CH₃, and —C₁₋₄alkyl; and R⁷ is selected from H,        —C₁₋₆alkyl, —[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,        —CH₂OC(O)CHR^(d)—NH₂, —CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl,        —CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,        —CH₂CH(NH₂)C(O)OCH₃, —C₂₋₄alkylene-N(CH₃)₂,        —C₀₋₆alkylenemorpholinyl, and

or

(v) X is

R^(a) is selected from Cl and F and R^(b) is H; or R^(a) is H and R^(b)is selected from Cl, F, —CH₃, and —CN; or R^(a) is F and R^(b) is Cl; R²is selected from H, —C₁₋₆alkyl, —(CH₂)₂₋₃OR^(e), and—(CH₂)₂₋₃NR^(e)R^(e); R³ is selected from —OH, —OCH₃, —OCH₂CH₃, and—C₁₋₄alkyl; R⁴ is selected from H, —C₁₋₆alkyl, and phenyl; and R⁷ isselected from H, —C₁₋₆alkyl, —[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CH₂OC(O)CHR^(d)—NH₂, —CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—CH₂CH(NH₂)C(O)OCH₃, —C₂₋₄alkylene-N(CH₃)₂, —C₀₋₆alkylenemorpholinyl,and

or

(vi) X is

R^(a) is selected from Cl and F and R^(b) is H; or R^(a) is H and R^(b)is selected from Cl, F, —CH₃, and —CN; or R^(a) is F and R^(b) is Cl; R²is selected from H, —C₁₋₆alkyl, —(CH₂)₂₋₃OR^(e), and—(CH₂)₂₋₃NR^(e)R^(e); R⁴ is selected from H, —C₁₋₆alkyl, and phenyl; andR⁷ is selected from H, —C₁₋₆alkyl, —[(CH₂)₂O]₁₋₃CH₃,—CHR^(c)OC(O)—C₁₋₄alkyl, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, —CHR^(c)OC(O)O—C₂₋₄alkyl,—CHR^(c)OC(O)O-cyclohexyl, —CH₂CH(NH₂)C(O)OCH₃, —C₂₋₄alkylene-N(CH₃)₂,—C₀₋₆alkylenemorpholinyl, and

or

(vii) X is

R^(a) is selected from Cl and F and R^(b) is H; or R^(a) is H and R^(b)is selected from Cl, F, —CH₃, and —CN; or R^(a) is F and R^(b) is Cl; R²is selected from H, —C₁₋₆alkyl, —(CH₂)₂₋₃OR^(e), and—(CH₂)₂₋₃NR^(e)R^(e); and R⁷ is selected from H, —C₁₋₆alkyl,—[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, —CHR^(c)OC(O)O—C₂₋₄alkyl,—CHR^(c)OC(O)O-cyclohexyl, —CH₂CH(NH₂)C(O)OCH₃, —C₂₋₄alkylene-N(CH₃)₂,—C₀₋₆alkylenemorpholinyl, and

or

(viii) X is

R^(a) is selected from Cl and F and R^(b) is H; or R^(a) is H and R^(b)is selected from Cl, F, —CH₃, and —CN; or R^(a) is F and R^(b) is Cl; R²is selected from H, —C₁₋₆alkyl, —(CH₂)₂₋₃OR^(e), and—(CH₂)₂₋₃NR^(e)R^(e); and R⁷ is selected from H, —C₁₋₆alkyl,—[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, —CHR^(c)OC(O)O—C₂₋₄alkyl,—CHR^(c)OC(O)O-cyclohexyl, —CH₂CH(NH₂)C(O)OCH₃, —C₂₋₄alkylene-N(CH₃)₂,—C₀₋₆alkylenemorpholinyl, and

where each R^(c) is independently H or —C₁₋₃alkyl; each R^(d) isindependently H, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) isindependently H or —CH₃; or a pharmaceutically acceptable salt thereof.

The present invention provides compounds which are metabolized in vivoto compounds that have been found to possess neprilysin (NEP) enzymeinhibition activity. Accordingly, compounds of the invention areexpected to be useful and advantageous as therapeutic agents fortreating patients suffering from a disease or disorder that is treatedby inhibiting the NEP enzyme or by increasing the levels of its peptidesubstrates. Thus, one aspect of the invention relates to a method oftreating hypertension, heart failure, or renal disease, comprisingadministering to a patient a therapeutically effective amount of acompound of the invention.

Another aspect of the invention relates to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a compound of theinvention.

Yet another aspect of the invention relates to processes andintermediates useful for preparing compounds of the invention. Anotheraspect of the invention relates to a process of preparing apharmaceutically acceptable salt of a compound of formula I, comprisingcontacting a compound of formula I in free acid or base form with apharmaceutically acceptable base or acid. In other aspects, theinvention relates to products prepared by any of the processes describedherein, as well as novel intermediates used in such process.

Yet another aspect of the invention relates to the use of a compound offormula I or a pharmaceutically acceptable salt thereof, for themanufacture of a medicament, especially for the manufacture of amedicament useful for treating hypertension, heart failure, or renaldisease. Another aspect of the invention relates to use of a compound ofthe invention for inhibiting a NEP enzyme in a mammal. Still anotheraspect of the invention relates to the use of a compound of theinvention as a research tool. Other aspects and embodiments of theinvention are disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

When describing the compounds, compositions, methods and processes ofthe invention, the following terms have the following meanings unlessotherwise indicated. Additionally, as used herein, the singular forms“a,” “an,” and “the” include the corresponding plural forms unless thecontext of use clearly dictates otherwise. The terms “comprising”,“including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements. Allnumbers expressing quantities of ingredients, properties such asmolecular weight, reaction conditions, and so forth used herein are tobe understood as being modified in all instances by the term “about,”unless otherwise indicated. Accordingly, the numbers set forth hereinare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At least, and not as anattempt to limit the application of the doctrine of equivalents to thescope of the claims, each number should at least be construed in lightof the reported significant digits and by applying ordinary roundingtechniques.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms and include, for example,—C₁₋₆alkyl, meaning an alkyl group having from 1 to 6 carbon atoms wherethe carbon atoms are in any acceptable configuration. Representativealkyl groups include, by way of example, methyl, ethyl, n-propyl,isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, n-pentyl, n-hexyl, andthe like.

As used herein, the phrase “of the formula” or “having the formula” or“having the structure” is not intended to be limiting and is used in thesame way that the term “comprising” is commonly used. For example, ifone structure is depicted, it is understood that all stereoisomer andtautomer forms are encompassed, unless stated otherwise.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise unacceptable when used in the invention. Forexample, the term “pharmaceutically acceptable carrier” refers to amaterial that can be incorporated into a composition and administered toa patient without causing unacceptable biological effects or interactingin an unacceptable manner with other components of the composition. Suchpharmaceutically acceptable materials typically have met the requiredstandards of toxicological and manufacturing testing, and include thosematerials identified as suitable inactive ingredients by the U.S. Foodand Drug administration.

The term “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (for example, salts having acceptable mammalian safetyfor a given dosage regime). However, it is understood that the saltscovered by the invention are not required to be pharmaceuticallyacceptable salts, such as salts of intermediate compounds that are notintended for administration to a patient. Pharmaceutically acceptablesalts can be derived from pharmaceutically acceptable inorganic ororganic bases and from pharmaceutically acceptable inorganic or organicacids. In addition, when a compound of formula I contains both a basicmoiety, such as an amine, pyridine or imidazole, and an acidic moietysuch as a carboxylic acid or tetrazole, zwitterions may be formed andare included within the term “salt” as used herein. Salts derived frompharmaceutically acceptable inorganic bases include ammonium, calcium,copper, ferric, ferrous, lithium, magnesium, manganic, manganous,potassium, sodium, and zinc salts, and the like. Salts derived frompharmaceutically acceptable organic bases include salts of primary,secondary and tertiary amines, including substituted amines, cyclicamines, naturally-occurring amines and the like, such as arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. Salts derived frompharmaceutically acceptable inorganic acids include salts of boric,carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric orhydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Saltsderived from pharmaceutically acceptable organic acids include salts ofaliphatic hydroxyl acids (for example, citric, gluconic, glycolic,lactic, lactobionic, malic, and tartaric acids), aliphaticmonocarboxylic acids (for example, acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (for example, aspartic andglutamic acids), aromatic carboxylic acids (for example, benzoic,p-chlorobenzoic, diphenylacetic, gentisic, hippuric, and triphenylaceticacids), aromatic hydroxyl acids (for example, o-hydroxybenzoic,p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylic and3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic acids(for example, fumaric, maleic, oxalic and succinic acids), glucoronic,mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids(for example, benzenesulfonic, camphosulfonic, edisylic, ethanesulfonic,isethionic, methanesulfonic, naphthalenesulfonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic andp-toluenesulfonic acids), xinafoic acid, and the like.

As used herein, the term “prodrug” is intended to mean an inactive (orsignificantly less active) precursor of a drug that is converted intoits active form in the body under physiological conditions, for example,by normal metabolic processes. Such compounds may not necessarilypossess pharmacological activity at NEP, but may be administered orallyor parenterally and thereafter metabolized in the body to form acompound that is pharmacologically active at NEP.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need thereof, thatis, the amount of drug needed to obtain the desired therapeutic effect.For example, a therapeutically effective amount for treatinghypertension is an amount of compound needed to, for example, reduce,suppress, eliminate, or prevent the symptoms of hypertension, or totreat the underlying cause of hypertension. In one embodiment, atherapeutically effective amount is that amount of drug needed to reduceblood pressure or the amount of drug needed to maintain normal bloodpressure. On the other hand, the term “effective amount” means an amountsufficient to obtain a desired result, which may not necessarily be atherapeutic result. For example, when studying a system comprising a NEPenzyme, an “effective amount” may be the amount needed to inhibit theenzyme.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as hypertension) in apatient, such as a mammal (particularly a human) that includes one ormore of the following: (a) preventing the disease or medical conditionfrom occurring, i.e., preventing the reoccurrence of the disease ormedical condition or prophylactic treatment of a patient that ispre-disposed to the disease or medical condition; (b) ameliorating thedisease or medical condition, i.e., eliminating or causing regression ofthe disease or medical condition in a patient; (c) suppressing thedisease or medical condition, i.e., slowing or arresting the developmentof the disease or medical condition in a patient; or (d) alleviating thesymptoms of the disease or medical condition in a patient. For example,the term “treating hypertension” would include preventing hypertensionfrom occurring, ameliorating hypertension, suppressing hypertension, andalleviating the symptoms of hypertension (for example, lowering bloodpressure). The term “patient” is intended to include those mammals, suchas humans, that are in need of treatment or disease prevention or thatare presently being treated for disease prevention or treatment of aspecific disease or medical condition, as well as test subjects in whichthe crystalline compound is being evaluated or being used in an assay,for example an animal model.

All other terms used herein are intended to have their ordinary meaningas understood by those of ordinary skill in the art to which theypertain.

The compounds of the invention contain one or more chiral centers andtherefore, these compounds may be prepared and used in variousstereoisomeric forms. In some embodiments, in order to optimize thetherapeutic activity of the compounds of the invention, e.g., to treathypertension, it may be desirable that the carbon atoms have aparticular (R,R), (S,S), (S,R), or (R,S) configuration or are enrichedin a stereoisomeric form having such configuration. In otherembodiments, the compounds of the invention are present as racemicmixtures. Accordingly, the invention also relates to racemic mixtures,pure stereoisomers (e.g., enantiomers and diastereoisomers),stereoisomer-enriched mixtures, and the like unless otherwise indicated.When a chemical structure is depicted herein without anystereochemistry, it is understood that all possible stereoisomers areencompassed by such structure. Similarly, when a particular stereoisomeris shown or named herein, it will be understood by those skilled in theart that minor amounts of other stereoisomers may be present in thecompositions of the invention unless otherwise indicated, provided thatthe utility of the composition as a whole is not eliminated by thepresence of such other isomers. Individual stereoisomers may be obtainedby numerous methods that are well known in the art, including chiralchromatography using a suitable chiral stationary phase or support, orby chemically converting them into diastereoisomers, separating thediastereoisomers by conventional means such as chromatography orrecrystallization, then regenerating the original stereoisomer.

Additionally, where applicable, all cis-trans or E/Z isomers (geometricisomers), tautomeric forms and topoisomeric forms of the compounds ofthe invention are included within the scope of the invention unlessotherwise specified. For example, although a formula is depicted as:

it is understood that the compound may also exist in a tautomeric formsuch as:

and that both forms are covered by the invention. It is also understoodthat one tautomer may be predominant.

The compounds of the invention, as well as those compounds used in theirsynthesis, may also include isotopically-labeled compounds, that is,where one or more atoms have been enriched with atoms having an atomicmass different from the atomic mass predominately found in nature.Examples of isotopes that may be incorporated into the compounds offormula I, for example, include, but are not limited to, ²H, ³H, ¹³C,¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ³⁶Cl, and ¹⁸F. Of particular interest arecompounds of formula I enriched in tritium or carbon-14 which can beused, for example, in tissue distribution studies; compounds of theinvention enriched in deuterium especially at a site of metabolismresulting, for example, in compounds having greater metabolic stability;and compounds of formula I enriched in a positron emitting isotope, suchas ¹¹C, ¹⁸F, ¹⁵O and ¹³N, which can be used, for example, in PositronEmission Topography (PET) studies.

The nomenclature used herein to name the compounds of the invention isillustrated in the Examples herein. This nomenclature has been derivedusing the commercially available AutoNom software (MDL, San Leandro,Calif.).

U.S. Patent Publication No. 2012/0213806 specifically discloses(2S,4R)-5-biphenyl-4-yl-2-hydroxymethyl-2-methyl-4-[(3H-[1,2,3]triazole-4-carbonyl)amino]-pentanoicacid, which is represented by formula I′ (where R^(a) and R^(b) are H):

Compounds such as this can exist in a tautomer form, for example, as(2S,4R)-5-biphenyl-4-yl-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicacid. In one embodiment, this compound is referred to as the active formand is administered as a prodrug, which is metabolized in vivo to formthe compound of formula I′. U.S. Patent Publication No. 2012/0213806also discloses certain prodrugs of the compound of formula I′ such asthe ethyl ester, propyl ester, isopropyl ester, butyl ester, isobutylester, 3-methylbutyl ester, pentyl ester, medoxomil ester,2-morpholin-4-ylethyl ester, 2-morpholin-4-yl-2-oxo-ethyl ester,2-methoxyethyl ester, 2-(2-methoxyethoxy)ethyl ester,2-methanesulfonylethyl ester, 2-dimethylaminoethyl ester,2-piperidin-1-ylethyl ester, indan-5-yl ester, oxetan-3-yl ester,dimethylcarbamoylmethyl ester, methoxycarbonyl-methyl ester,acetoxymethyl ester, butyryloxymethyl ester, benzyloxycarbonylmethylester, 2-(2-oxopyrrolidin-1-yl)ethyl ester, ethoxycarbonyloxymethylester, benzyl ester, (S)-2-amino-3-methyl-butyryloxymethyl ester,(S)-2-methoxycarbonylamino-3-methyl-butyryloxymethyl ester,(R)-1-cyclohexyloxycarbonyloxyethyl ester,(S)-1-cyclohexyloxycarbonyloxyethyl ester; and5-methyl-2-oxo-[1,3]dioxol-4-ylmethyl ester prodrugs.

One aspect of the invention relates to other prodrugs and variants ofthe compound of formula I′. These compounds are compounds of formula I,where X is:

These compounds are represented by formula IIa or IIb:

In one embodiment of the compounds of formula IIa and IIb, R^(a) andR^(b) are H; R² is H; and R⁷ is selected from —CH₂CF₂CH₃, —CH₂CF₂CF₃,—(CH₂)₅CH₃, —(CH₂)₆CH₃, and

or R² is —C₁₋₆alkyl or —C(O)—C₁₋₆alkyl, and R⁷ is H. In one specificembodiment, R² is H and R⁷ is selected from —CH₂CF₂CH₃, —CH₂CF₂CF₃,—(CH₂)₅CH₃, —(CH₂)₆CH₃, and

orR² selected from —CH₃, —CH₂CH₃, —C(O)CH₃, —C(O)CH(CH₃)₂, and—C(O)CH₂CH(CH₃)₂; and R⁷ is H.

In another embodiment of the compounds of formula IIa and IIb, R^(a) isselected from —CH₃, —OCH₃, and Cl and R^(b) is H; or R^(a) is selectedfrom H, —CH₃, Cl, and F, and R^(b) is Cl; or R^(a) is H and R^(b) isselected from —CH₃ and —CN; R² is selected from H, —C₁₋₆alkyl,—(CH₂)₂₋₃OR^(e), and —(CH₂)₂₋₃NR^(e)R^(e); and R⁷ is selected from H,—C₁₋₆alkyl, —[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CH₂OC(O)CHR^(d)—NH₂, —CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—CH₂CH(NH₂)C(O)OCH₃, —C₂₋₄alkylene-N(CH₃)₂, —C₀₋₆alkylenemorpholinyl,and

where each R^(c) is independently H or —C₁₋₃alkyl; each R^(d) isindependently H, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) isindependently H or —CH₃. In one specific embodiment, R^(a) is selectedfrom —CH₃, —OCH₃, and Cl and R^(b) is H; or R^(a) is selected from H,—CH₃, Cl, and F, and R^(b) is Cl; or R^(a) is H and R^(b) is selectedfrom —CH₃ and —CN; R² is selected from H, —C₁₋₆alkyl (e.g., —CH₃,—CH₂CH₃, —CH(CH₃), or —(CH₂)₄CH₃), and —(CH₂)₂₋₃OR^(e) where R^(e) is H(e.g., —(CH₂)₂OH and —(CH₂)₃OH) or —CH₃ (e.g., —(CH₂)₂OCH₃); and R⁷ isH.

U.S. Patent Publication No. 2012/0213806 also discloses compounds offormula I′, where R^(a) is H and R^(b) is F,(2S,4R)-5-(3′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(3H-[1,2,3]triazole-4-carbonyl)amino]pentanoicacid, and where R^(a) is F and R^(b) is H,(2S,4R)-5-(2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(3H-[1,2,3]triazole-4-carbonyl)amino]pentanoicacid. Thus, another aspect of the invention relates to prodrugs of suchcompounds. Therefore, in another embodiment of the compounds of formulaIIa and IIb, R^(a) is H and R^(b) is F; or R^(a) is F and R^(b) is H; R²is selected from H, —C₁₋₆alkyl, —(CH₂)₂₋₃OR^(e), and—(CH₂)₂₋₃NR^(e)R^(e); and R⁷ is selected from —C₁₋₆alkyl,—[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, —CHR^(c)OC(O)O—C₂₋₄alkyl,—CHR^(c)OC(O)O— cyclohexyl, —CH₂CH(NH₂)C(O)OCH₃, —C₂₋₄alkylene-N(CH₃)₂,—C₀₋₆alkylenemorpholinyl, and

where each R^(c) is independently H or —C₁₋₃alkyl; each R^(d) isindependently H, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) isindependently H or —CH₃.

Another aspect of the invention relates to compounds of formula I, whereX is:

These compounds are represented by formula III:

In one embodiment of the compounds of formula III, R^(a) is Cl and R^(b)is H; or R^(a) is H and R^(b) is selected from Cl, F, —CH₃, and —CN; orR^(a) is F and R^(b) is Cl; R² is selected from H, —C₁₋₆alkyl,—(CH₂)₂₋₃OR^(e), and —(CH₂)₂₋₃NR^(e)R^(e); R⁴ is selected from —OH,—OCH₃, —OCH₂CH₃, and —C₁₋₄alkyl; and R⁷ is selected from H, —C₁₋₆alkyl,—[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, —CHR^(c)OC(O)O—C₂₋₄alkyl,—CHR^(c)OC(O)O-cyclohexyl, —CH₂CH(NH₂)C(O)OCH₃, —C₂₋₄alkylene-N(CH₃)₂,—C₀₋₆alkylenemorpholinyl, and

where each R^(c) is independently H or —C₁₋₃alkyl; each R^(d) isindependently H, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) isindependently H or —CH₃. In one specific embodiment, R^(a) is F, R^(b)is Cl, R² is H, R⁴ is —OCH₃ or —OCH₂CH₃, and R⁷ is H.

U.S. Patent Publication No. 2012/0213806 discloses a compound of formulaIII, where R^(a) is F, R^(b) is H, R² is H, and R⁷ is H,(2S,4R)-5-(2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-4-[(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)amino]-2-methylpentanoicacid. Thus, another aspect of the invention relates to prodrugs of thiscompound. Therefore, in another embodiment of the compounds of formulaIII, R^(a) is F and R^(b) is H; R² is H; R⁴ is —OH; and R⁷ is selectedfrom —C₁₋₆alkyl, —[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CH₂OC(O)CHR^(d)—NH₂, —CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—CH₂CH(NH₂)C(O)OCH₃, —C₂₋₄alkylene-N(CH₃)₂, —C₀₋₆alkylenemorpholinyl,and

where each R^(c) is independently H or —C₁₋₃alkyl; and each R^(d) isindependently H, —CH₃, —CH(CH₃)₂, phenyl, or benzyl.

Another aspect of the invention relates to compounds of formula I, whereX is:

These compounds are represented by formula IVa or IVb:

In one embodiment of the compounds of formula IVa and IVb, R^(a) is Cland R^(b) is H; or R^(a) is H and R^(b) is selected from Cl, F, —CH₃,and —CN; or R^(a) is F and R^(b) is Cl; R² is selected from H,—C₁₋₆alkyl, —(CH₂)₂₋₃OR^(e), and —(CH₂)₂₋₃NR^(e)R^(e); and R⁷ isselected from H, —C₁₋₆alkyl, —[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CH₂OC(O)CHR^(d)—NH₂, —CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—CH₂CH(NH₂)C(O)OCH₃, —C₂₋₄alkylene-N(CH₃)₂, —C₀₋₆alkylenemorpholinyl,and

where each R^(c) is independently H or —C₁₋₃alkyl; each R^(d) isindependently H, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) isindependently H or —CH₃. In one specific embodiment, R^(a) is F, R^(b)is Cl, R² is H, and R⁷ is H.

U.S. Patent Publication No. 2012/0213806 discloses a compound of formulaIVa, where R^(a) is F, R^(b) is H, R² is H, and R⁷ is H,(2S,4R)-5-(2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(2-oxo-2,3-dihydrooxazole-4-carbonyl)amino]pentanoicacid, and a compound of formula IVb, where R^(a) is F, R^(b) is H, R² isH, and R⁷ is H,(2S,4R)-5-(2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(2-oxo-2,3-dihydrooxazole-5-carbonyl)amino]pentanoicacid. Thus, another aspect of the invention relates to prodrugs of thesecompounds. Therefore, in another embodiment of the compounds of formulaIVa and IVb, R^(a) is F and R^(b) is H; R² is H; and R⁷ is selected from—C₁₋₆alkyl, —[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CH₂OC(O)CHR^(d)—NH₂, —CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—CH₂CH(NH₂)C(O)OCH₃, —C₂₋₄alkylene-N(CH₃)₂, —C₀₋₆alkylenemorpholinyl,and

where each R^(c) is independently H or —C₁₋₃alkyl; and each R^(d) isindependently H, —CH₃, —CH(CH₃)₂, phenyl, or benzyl.

U.S. Patent Publication No. 2012/0213806 specifically discloses(2S,4R)-5-biphenyl-4-yl-4-[(3-hydroxyisoxazole-5-carbonyl)amino]-2-hydroxymethyl-2-methylpentanoicacid, which is represented by formula V′ (where R^(a) and R^(b) are Hand R³ is —OH):

U.S. Patent Publication No. 2012/0213806 also discloses certain prodrugsof the compound of formula V′ such as the ethyl ester. One aspect of theinvention relates to other prodrugs and variants of the compound offormula V′. These compounds are compounds of formula I, where X is:

These compounds are represented by formula V:

In one embodiment of the compounds of formula V, R^(a) and R^(b) are H;R² is selected from —C₁₋₆alkyl, —(CH₂)₂₋₃OR^(e), and—(CH₂)₂₋₃NR^(e)R^(e); R³ is selected from —OH, —OCH₃, —OCH₂CH₃, and—C₁₋₄alkyl; and R⁷ is H; where each R^(e) is independently H or —CH₃. Inone specific embodiment of the compounds of formula V, R^(a) and R^(b)are H, R² is —CH₃, R³ is —OH or —OCH₃, and R⁷ is H.

In another embodiment of the compounds of formula V, R^(a) is selectedfrom Cl and F and R^(b) is H; or R^(a) is H and R^(b) is selected fromCl, F, —CH₃, and —CN; or R^(a) is F and R^(b) is Cl; R² is selected fromH, —C₁₋₆alkyl, —(CH₂)₂₋₃OR^(e), and —(CH₂)₂₋₃NR^(e)R^(e); R³ is selectedfrom —OH, —OCH₃, —OCH₂CH₃, and —C₁₋₄alkyl; and R⁷ is selected from H,—C₁₋₆alkyl, —[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CH₂OC(O)CHR^(d)—NH₂, —CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—CH₂CH(NH₂)C(O)OCH₃, —C₂₋₄alkylene-N(CH₃)₂, —C₀₋₆alkylenemorpholinyl,and

where each R^(c) is independently H or —C₁₋₃alkyl; each R^(d) isindependently H, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) isindependently H or —CH₃. In one specific embodiment of the compounds offormula V, R^(a) is H, R^(b) is Cl, R² is H, —CH₃, —CH₂CH₃ or —(CH₂)₂OH,R³ is —OH or —OCH₃, and R⁷ is H; or R^(a) is F, R^(b) is Cl, R² is H or—C₁₋₆alkyl (e.g., —CH₃ or —CH₂CH₃), R³ is —OH, —OCH₃ or —C₁₋₄alkyl(e.g., —CH₂CH₃, —(CH₂)₂CH₃, or —CH₂CH(CH₃)₂), and R⁷ is H.

Another aspect of the invention relates to compounds of formula I, whereX is:

These compounds are represented by formula VIa or VIb:

In one embodiment of the compounds of formula VI, R^(a) is selected fromCl and F and R^(b) is H; or R^(a) is H and R^(b) is selected from Cl, F,—CH₃, and —CN; or R^(a) is F and R^(b) is Cl; R² is selected from H,—C₁₋₆alkyl, —(CH₂)₂₋₃OR^(e), and —(CH₂)₂₋₃NR^(e)R^(e); R³ is selectedfrom —OH, —OCH₃, —OCH₂CH₃, and —C₁₋₄alkyl; R⁴ is selected from H,—C₁₋₆alkyl, and phenyl; and R⁷ is selected from H, —C₁₋₆alkyl,—[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, —CHR^(c)OC(O)O—C₂₋₄alkyl,—CHR^(c)OC(O)O-cyclohexyl, —CH₂CH(NH₂)C(O)OCH₃, —C₂₋₄alkylene-N(CH₃)₂,—C₀₋₆alkylenemorpholinyl, and

where each R^(c) is independently H or —C₁₋₃alkyl; each R^(d) isindependently H, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) isindependently H or —CH₃. In one specific embodiment of the compounds offormula VIa and VIb, R^(a) is H or F; R^(b) is Cl; R² is H or —C₁₋₆alkyl(e.g., —CH₃); R³ is —OCH₃, —OCH₂CH₃ or —C₁₋₄alkyl (e.g., —CH(CH₃)₂ or—CH₂CH(CH₃)₂)); R⁴, if present, is H; and R⁷ is H.

Another aspect of the invention relates to compounds of formula I, whereX is:

These compounds are represented by formula VIIa or VIIb:

In one embodiment of the compounds of formula VII, R^(a) is selectedfrom Cl and F and R^(b) is H; or R^(a) is H and R^(b) is selected fromCl, F, —CH₃, and —CN; or R^(a) is F and R^(b) is Cl; R² is selected fromH, —C₁₋₆alkyl, —(CH₂)₂₋₃OR^(e), and —(CH₂)₂₋₃NR^(e)R^(e); R⁴ is selectedfrom H, —C₁₋₆alkyl, and phenyl; and R⁷ is selected from H, —C₁₋₆alkyl,—[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl, —CH₂OC(O)CHR^(d)—NH₂,—CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl, —CHR^(c)OC(O)O—C₂₋₄alkyl,—CHR^(c)OC(O)O-cyclohexyl, —CH₂CH(NH₂)C(O)OCH₃, —C₂₋₄alkylene-N(CH₃)₂,—C₀₋₆alkylenemorpholinyl, and

where each R^(c) is independently H or —C₁₋₃alkyl; each R^(d) isindependently H, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) isindependently H or —CH₃. In one specific embodiment of the compounds offormula VIIa and VIIb, R^(a) is F, R^(b) is Cl, R² is H or —C₁₋₆alkyl(e.g., —CH₃ or —CH₂CH₃), R⁴, if present, is H, and R⁷ is H.

Another aspect of the invention relates to compounds of formula I, whereX is:

These compounds are represented by formula VIII:

In one embodiment of the compounds of formula VIII, R^(a) is selectedfrom Cl and F and R^(b) is H; or R^(a) is H and R^(b) is selected fromCl, F, —CH₃, and —CN; or R^(a) is F and R^(b) is Cl; R² is selected fromH, —C₁₋₆alkyl, —(CH₂)₂₋₃OR^(e), and —(CH₂)₂₋₃NR^(e)R^(e); and R⁷ isselected from H, —C₁₋₆alkyl, —[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CH₂OC(O)CHR^(d)—NH₂, —CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O-cyclohexyl,—CH₂CH(NH₂)C(O)OCH₃, —C₂₋₄alkylene-N(CH₃)₂, —C₀₋₆alkylenemorpholinyl,and

where each R^(c) is independently H or —C₁₋₃alkyl; each R^(d) isindependently H, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) isindependently H or —CH₃.

Another aspect of the invention relates to compounds of formula I, whereX is:

These compounds are represented by formula IX:

In one embodiment of the compounds of formula IX, R^(a) is selected fromCl and F and R^(b) is H; or R^(a) is H and R^(b) is selected from Cl, F,—CH₃, and —CN; or R^(a) is F and R^(b) is Cl; R² is selected from H,—C₁₋₆alkyl, —(CH₂)₂₋₃OR^(e), and —(CH₂)₂₋₃NR^(e)R^(e); and R⁷ isselected from H, —C₁₋₆alkyl, —[(CH₂)₂O]₁₋₃CH₃, —CHR^(c)OC(O)—C₁₋₄alkyl,—CH₂OC(O)CHR^(d)—NH₂, —CH₂OC(O)CHR^(d)—NHC(O)O—C₁₋₆alkyl,—CHR^(c)OC(O)O—C₂₋₄alkyl, —CHR^(c)OC(O)O— cyclohexyl,—CH₂CH(NH₂)C(O)OCH₃, —C₂₋₄alkylene-N(CH₃)₂, —C₀₋₆alkylenemorpholinyl,and

where each R^(c) is independently H or —C₁₋₃alkyl; each R^(d) isindependently H, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; and each R^(e) isindependently H or —CH₃.

General Synthetic Procedures

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods, the proceduresset forth in the Examples, or by using other methods, reagents, andstarting materials that are known to those of ordinary skill in the art.Although the following procedures may illustrate a particular embodimentof the invention, it is understood that other embodiments of theinvention can be similarly prepared using the same or similar methods orby using other methods, reagents and starting materials known to thoseof ordinary skill in the art. It will also be appreciated that wheretypical or preferred process conditions (for example, reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. In some instances, reactions were conducted at roomtemperature and no actual temperature measurement was taken. It isunderstood that room temperature can be taken to mean a temperaturewithin the range commonly associated with the ambient temperature in alaboratory environment, and will typically be in the range of about 18°C. to about 30° C. In other instances, reactions were conducted at roomtemperature and the temperature was actually measured and recorded.While optimum reaction conditions will typically vary depending onvarious reaction parameters such as the particular reactants, solventsand quantities used, those of ordinary skill in the art can readilydetermine suitable reaction conditions using routine optimizationprocedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions and reagents for protection anddeprotection of such functional groups are well-known in the art.Protecting groups other than those illustrated in the proceduresdescribed herein may be used, if desired. For example, numerousprotecting groups, and their introduction and removal, are described inT. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis,Fourth Edition, Wiley, New York, 2006, and references cited therein.

Carboxy-protecting groups are suitable for preventing undesiredreactions at a carboxy group, and examples include, but are not limitedto, methyl, ethyl, t-butyl, benzyl (Bn), p-methoxybenzyl (PMB),9-fluorenylmethyl (Fm), trimethylsilyl (TMS), t-butyldimethylsilyl(TBDMS), diphenylmethyl (benzhydryl, DPM) and the like. Amino-protectinggroups are suitable for preventing undesired reactions at an aminogroup, and examples include, but are not limited to, t-butoxycarbonyl(BOC), trityl (Tr), benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl(Fmoc), formyl, trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS), andthe like. Hydroxyl-protecting groups are suitable for preventingundesired reactions at a hydroxyl group, and examples include, but arenot limited to C₁₋₆alkyls, silyl groups including triC₁₋₆alkylsilylgroups, such as trimethylsilyl (TMS), triethylsilyl (TES), andtert-butyldimethylsilyl (TBDMS); esters (acyl groups) includingC₁₋₆alkanoyl groups, such as formyl, acetyl, and pivaloyl, and aromaticacyl groups such as benzoyl; arylmethyl groups such as benzyl (Bn),p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl(benzhydryl, DPM); and the like.

Standard deprotection techniques and reagents are used to remove theprotecting groups, and may vary depending upon which group is used. Forexample, sodium or lithium hydroxide is commonly used when thecarboxy-protecting group is methyl, an acid such as TFA or HCl (e.g.,4.0 M HCl in 1,4-dioxane) is commonly used when the carboxy-protectinggroup is ethyl or t-butyl, and H₂/Pd/C may be used when thecarboxy-protecting group is benzyl. A BOC amino-protecting group can beremoved using an acidic reagent such as TFA in DCM or HCl in1,4-dioxane, while a Cbz amino-protecting group can be removed byemploying catalytic hydrogenation conditions such as H₂ (1 atm) and 10%Pd/C in an alcoholic solvent (“H₂/Pd/C”). H₂/Pd/C is commonly used whenthe hydroxyl-protecting group is benzyl, while NaOH is commonly usedwhen the hydroxyl-protecting group is an acyl group.

Leaving groups are functional groups or atoms that can be displaced byanother functional group or atom in a substitution reaction, such as anucleophilic substitution reaction. By way of example, representativeleaving groups include chloro, bromo and iodo groups; sulfonic estergroups, such as mesylate, tosylate, brosylate, nosylate and the like;and acyloxy groups, such as acetoxy, trifluoroacetoxy and the like.

Suitable bases for use in these schemes include, by way of illustrationand not limitation, potassium carbonate, calcium carbonate, sodiumcarbonate, triethylamine (Et₃N), pyridine,1,8-diazabicyclo-[5.4.0]undec-7-ene (DBU), N,N-diisopropylethylamine(DIPEA), 4-methylmorpholine, sodium hydroxide, potassium hydroxide,potassium t-butoxide, and metal hydrides.

Suitable inert diluents or solvents for use in these schemes include, byway of illustration and not limitation, tetrahydrofuran (THF),acetonitrile (MeCN), N,N-dimethylformamide (DMF), N,N-dimethylacetamide(DMA), dimethyl sulfoxide (DMSO), toluene, dichloromethane (DCM),chloroform (CHCl₃), carbon tetrachloride (CCl₄), 1,4-dioxane, methanol,ethanol, water, diethyl ether, acetone, and the like.

Suitable carboxylic acid/amine coupling reagents includebenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (HATU), 1,3-dicyclohexylcarbodiimide (DCC),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC),carbonyldiimidazole (CDI), 1-hydroxybenzotriazole (HOBt), and the like.Coupling reactions are conducted in an inert diluent in the presence ofa base such as DIPEA, and are performed under conventional amidebond-forming conditions.

All reactions are typically conducted at a temperature within the rangeof about −78 C to 100° C., for example at room temperature. Reactionsmay be monitored by use of thin layer chromatography (TLC), highperformance liquid chromatography (HPLC), and/or LCMS until completion.Reactions may be complete in minutes, or may take hours, typically from1-2 hours and up to 48 hours. Upon completion, the resulting mixture orreaction product may be further treated in order to obtain the desiredproduct. For example, the resulting mixture or reaction product may besubjected to one or more of the following procedures: concentrating orpartitioning (for example, between EtOAc and water or between 5% THF inEtOAc and 1M phosphoric acid); extraction (for example, with EtOAc,CHCl₃, DCM, chloroform); washing (for example, with saturated aqueousNaCl, saturated aqueous NaHCO₃, Na₂CO₃ (5%), CHCl₃ or 1M NaOH); drying(for example, over MgSO₄, over Na₂SO₄, or in vacuo); filtering;crystallizing (for example, from EtOAc and hexanes); being concentrated(for example, in vacuo); and/or purification (e.g., silica gelchromatography, flash chromatography, preparative HPLC, reversephase-HPLC, or crystallization).

By way of illustration, compounds of formula I, as well as their salts,can be prepared as shown in Schemes I-IV.

Scheme I is a transesterification reactions. Generally, this reactioninvolves reacting the ester with heat, the desired alcohol (HO—R⁷) and asuitable acid catalyst, for example hydrochloric acid. The HO—R⁷alcohols are either commercially available or can be prepared bytechniques that are known in the art or described herein. ExemplaryHO—R⁷ groups include HO—CH₂CF₂CH₃, HO—CH₂CF₂CF₃, and

Scheme II is a nucleophilic substitution reaction, where L is a suitableleaving group. Generally, this reaction is conducted in the presence ofa suitable base such as triethylamine in a suitable inert diluent orsolvent such as acetone. The L-R⁷ compound is either commerciallyavailable or can be prepared by techniques that are known in the art ordescribed herein.

Scheme III is a nucleophilic substitution reaction, where L is asuitable leaving group. Generally, this reaction is conducted in thepresence of a suitable base such as N,N-diisopropylethylamine in asuitable inert diluent or solvent such as dichloromethane. The L-R²compound is either commercially available or can be prepared bytechniques that are known in the art or described herein. Exemplary L-R²compounds include Cl—C(O)—CH₃, Cl—C(O)—CH(CH₃)₂, andCl—C(O)—CH₂CH(CH₃)₂.

Scheme IV is a coupling reaction, where P is H or a suitableamino-protecting group. When P is an amino protecting group, the processfurther comprises deprotecting the compound, before or in situ with thecoupling step. Exemplary coupling reagents include HATU and HOBt withEDC. Generally, this reaction is conducted in the presence of a basesuch as DIPEA or 4-methylmorpholine, and an inert diluent or solventssuch as DMF or DMA. The carboxylic acid starting materials are generallycommercially available or can be prepared using procedures that areknown in the art.

By way of illustration, compounds of formulas II-X, as well as theirsalts, can be prepared as shown in Scheme V.

(R)-3-(4-bromophenyl)-2-t-butoxycarbonylaminopropionic acid and thedesired halophenylboronic acid are combined with a palladium catalyst inan inert diluent in the presence of an a suitable base such as potassiumcarbonate or sodium carbonate. Exemplary halophenylboronic acids are2-fluorophenylboronic acid, 3-fluorophenylboronic acid,2-chlorophenylboronic acid, 3-chlorophenylboronic acid, and2-fluoro-5-chlorophenylboronic acid. Exemplary palladium catalystsinclude 1,1-bis(diphenylphosphino) ferrocene palladium chloride,dichlorobis(triphenylphosphine) palladium (II),bis(tri-t-butylphosphine) palladium(0), and tetrakis(triphenylphosphine)palladium(0).

Compound 1 is then converted to Compound 5 (where P is H or a suitableamino-protecting group) by a several step process, which is detailed inthe Examples section.

Finally, Compound 5 is coupled with the desired X group as describedabove in Scheme IV:

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of the invention orintermediates thereof are described in the Examples set forth below.

Utility

The compound of formula I′ has activity as a neprilysin inhibitor, andis expected to have therapeutic utility as a neprilysin inhibitor.Prodrugs of this compound, once metabolized in vivo, are expected tohave the same utility. Thus, when discussing the activity of thecompounds of the invention, it is understood that these prodrugs havethe expected activity once metabolized.

Exemplary assays include by way of illustration and not limitation,assays that measure NEP inhibition. Useful secondary assays includeassays to measure ACE inhibition and aminopeptidase P (APP) inhibition(e.g., as described in Sulpizio et al. (2005) JPET 315:1306-1313). Apharmacodynamic assay to assess the in vivo inhibitory potencies for ACEand NEP in anesthetized rats is described in Seymour et al. (1985)Hypertension 7(Suppl I):I-35-I-42 and Wigle et al. (1992) Can. JPhysiol. Pharmacol. 70:1525-1528), where ACE inhibition is measured asthe percent inhibition of the angiotensin I pressor response and NEPinhibition is measured as increased urinary cyclic guanosine 3′,5′-monophosphate (cGMP) output.

There are also many in vivo assays that can be used. The consciousspontaneously hypertensive rat (SHR) model is a renin dependenthypertension model. See for example, Intengan et al. (1999) Circulation100(22):2267-2275 and Badyal et al. (2003) Indian Journal ofPharmacology 35:349-362. The conscious desoxycorticosterone acetate-salt(DOCA-salt) rat model is a volume dependent hypertension model that isuseful for measuring NEP activity. See for example, Trapani et al.(1989) J. Cardiovasc. Pharmacol. 14:419-424, Intengan et al. (1999)Hypertension 34(4):907-913, and Badyal et al. (2003) supra). TheDOCA-salt model is particularly useful for evaluating the ability of atest compound to reduce blood pressure as well as to measure a testcompound's ability to prevent or delay a rise in blood pressure. TheDahl salt-sensive (DSS) hypertensive rat model is a model ofhypertension that is sensitive to dietary salt (NaCl), and is described,for example, in Rapp (1982) Hypertension 4:753-763. The ratmonocrotaline model of pulmonary arterial hypertension described, forexample, in Kato et al. (2008) J. Cardiovasc. Pharmacol. 51(1):18-23, isa reliable predictor of clinical efficacy for the treatment of pulmonaryarterial hypertension. Heart failure animal models include the DSS ratmodel for heart failure and the aorto-caval fistula model (AV shunt),the latter of which is described, for example, in Norling et al. (1996)J. Amer. Soc. Nephrol. 7:1038-1044. Other animal models, such as the hotplate, tail-flick and formalin tests, can be used to measure theanalgesic properties of a compound, as well as the spinal nerve ligation(SNL) model of neuropathic pain. See, for example, Malmberg et al.(1999) Current Protocols in Neuroscience 8.9.1-8.9.15. Other propertiesand utilities of the compounds can be demonstrated using various invitro and in vivo assays well known to those skilled in the art.

The compounds of the invention are expected to be useful for thetreatment and/or prevention of medical conditions responsive to NEPinhibition. Thus it is expected that patients suffering from a diseaseor disorder that is treated by inhibiting the NEP enzyme or byincreasing the levels of its peptide substrates, can be treated byadministering a therapeutically effective amount of a compound of theinvention. For example, by inhibiting NEP, the compound is expected topotentiate the biological effects of endogenous peptides that aremetabolized by NEP, such as the natriuretic peptides, bombesin,bradykinins, calcitonin, endothelins, enkephalins, neurotensin,substance P and vasoactive intestinal peptide. Thus, the compounds areexpected to have other physiological actions, for example, on the renal,central nervous, reproductive and gastrointestinal systems.

Cardiovascular Diseases

By potentiating the effects of vasoactive peptides like the natriureticpeptides and bradykinin, compounds of the invention are expected to findutility in treating and/or preventing medical conditions such ascardiovascular diseases. See, for example, Roques et al. (1993)Pharmacol. Rev. 45:87-146 and Dempsey et al. (2009) Amer. J. ofPathology 174(3):782-796. Cardiovascular diseases of particular interestinclude hypertension and heart failure. Hypertension includes, by way ofillustration and not limitation: primary hypertension, which is alsoreferred to as essential hypertension or idiopathic hypertension;secondary hypertension; hypertension with accompanying renal disease;severe hypertension with or without accompanying renal disease;pulmonary hypertension, including pulmonary arterial hypertension; andresistant hypertension. Heart failure includes, by way of illustrationand not limitation: congestive heart failure; acute heart failure;chronic heart failure, for example with reduced left ventricularejection fraction (also referred to as systolic heart failure) or withpreserved left ventricular ejection fraction (also referred to asdiastolic heart failure); and acute and chronic decompensated heartfailure, with or without accompanying renal disease. Thus, oneembodiment of the invention relates to a method for treatinghypertension, particularly primary hypertension or pulmonary arterialhypertension, comprising administering to a patient a therapeuticallyeffective amount of a compound of the invention.

For treatment of primary hypertension, the therapeutically effectiveamount is typically the amount that is sufficient to lower the patient'sblood pressure. This would include both mild-to-moderate hypertensionand severe hypertension. When used to treat hypertension, the compoundmay be administered in combination with other therapeutic agents such asaldosterone antagonists, angiotensin-converting enzyme inhibitors anddual-acting angiotensin-converting enzyme/neprilysin inhibitors,angiotensin-converting enzyme 2 (ACE2) activators and stimulators,angiotensin-II vaccines, anti-diabetic agents, anti-lipid agents,anti-thrombotic agents, AT₁ receptor antagonists and dual-acting AT₁receptor antagonist/neprilysin inhibitors, β₁-adrenergic receptorantagonists, dual-acting β-adrenergic receptor antagonist/α₁-receptorantagonists, calcium channel blockers, diuretics, endothelin receptorantagonists, endothelin converting enzyme inhibitors, neprilysininhibitors, natriuretic peptides and their analogs, natriuretic peptideclearance receptor antagonists, nitric oxide donors, non-steroidalanti-inflammatory agents, phosphodiesterase inhibitors (specificallyPDE-V inhibitors), prostaglandin receptor agonists, renin inhibitors,soluble guanylate cyclase stimulators and activators, and combinationsthereof. In one particular embodiment of the invention, a compound ofthe invention is combined with an AT₁ receptor antagonist, a diuretic, acalcium channel blocker, or a combination thereof, and used to treatprimary hypertension. In another particular embodiment of the invention,a compound of the invention is combined with an AT₁ receptor antagonist,and used to treat hypertension with accompanying renal disease.

For treatment of pulmonary arterial hypertension, the therapeuticallyeffective amount is typically the amount that is sufficient to lower thepulmonary vascular resistance. Other goals of therapy are to improve apatient's exercise capacity. For example, in a clinical setting, thetherapeutically effective amount can be the amount that improves apatient's ability to walk comfortably for a period of 6 minutes(covering a distance of approximately 20-40 meters). When used to treatpulmonary arterial hypertension the compound may be administered incombination with other therapeutic agents such as α-adrenergicantagonists, β₁-adrenergic receptor antagonists, β₂-adrenergic receptoragonists, angiotensin-converting enzyme inhibitors, anticoagulants,calcium channel blockers, diuretics, endothelin receptor antagonists,PDE-V inhibitors, prostaglandin analogs, selective serotonin reuptakeinhibitors, and combinations thereof. In one particular embodiment ofthe invention, a compound of the invention is combined with a PDE-Vinhibitor or a selective serotonin reuptake inhibitor and used to treatpulmonary arterial hypertension.

Another embodiment of the invention relates to a method for treatingheart failure, in particular congestive heart failure (including bothsystolic and diastolic congestive heart failure), comprisingadministering to a patient a therapeutically effective amount of acompound of the invention. Typically, the therapeutically effectiveamount is the amount that is sufficient to lower blood pressure and/orimprove renal functions. In a clinical setting, the therapeuticallyeffective amount can be the amount that is sufficient to improve cardiachemodynamics, like for instance reduction in wedge pressure, rightatrial pressure, filling pressure, and vascular resistance. In oneembodiment, the compound is administered as an intravenous dosage form.When used to treat heart failure, the compound may be administered incombination with other therapeutic agents such as adenosine receptorantagonists, advanced glycation end product breakers, aldosteroneantagonists, AT₁ receptor antagonists, β₁-adrenergic receptorantagonists, dual-acting β-adrenergic receptor antagonist/α₁-receptorantagonists, chymase inhibitors, digoxin, diuretics, endothelinconverting enzyme (ECE) inhibitors, endothelin receptor antagonists,natriuretic peptides and their analogs, natriuretic peptide clearancereceptor antagonists, nitric oxide donors, prostaglandin analogs, PDE-Vinhibitors, soluble guanylate cyclase activators and stimulators, andvasopressin receptor antagonists. In one particular embodiment of theinvention, a compound of the invention is combined with an aldosteroneantagonist, a β₁-adrenergic receptor antagonist, an AT₁ receptorantagonist, or a diuretic, and used to treat congestive heart failure.

Diarrhea

As NEP inhibitors, compounds of the invention are expected to inhibitthe degradation of endogenous enkephalins and thus such compounds mayalso find utility for the treatment of diarrhea, including infectiousand secretory/watery diarrhea. See, for example, Baumer et al. (1992)Gut 33:753-758; Farthing (2006) Digestive Diseases 24:47-58; andMarcais-Collado (1987) Eur. J Pharmacol. 144(2):125-132. When used totreat diarrhea, compounds of the invention may be combined with one ormore additional antidiarrheal treatments.

Renal Diseases

By potentiating the effects of vasoactive peptides like the natriureticpeptides and bradykinin, compounds of the invention are expected toenhance renal function (see Chen et al. (1999) Circulation100:2443-2448; Lipkin et al. (1997) Kidney Int. 52:792-801; and Dussauleet al. (1993) Clin. Sci. 84:31-39) and find utility in treating and/orpreventing renal diseases. Renal diseases of particular interest includediabetic nephropathy, chronic kidney disease, proteinuria, andparticularly acute kidney injury or acute renal failure (see Sharkovskaet al. (2011) Clin. Lab. 57:507-515 and Newaz et al. (2010) RenalFailure 32:384-390). When used to treat renal disease, the compound maybe administered in combination with other therapeutic agents such asangiotensin-converting enzyme inhibitors, AT₁ receptor antagonists, anddiuretics.

Preventative Therapy

By potentiating the effects of the natriuretic peptides, compounds ofthe invention are also expected to be useful in preventative therapy,due to the antihypertrophic and antifibrotic effects of the natriureticpeptides (see Potter et al. (2009) Handbook of Experimental Pharmacology191:341-366), for example in preventing the progression of cardiacinsufficiency after myocardial infarction, preventing arterialrestenosis after angioplasty, preventing thickening of blood vesselwalls after vascular operations, preventing atherosclerosis, andpreventing diabetic angiopathy.

Glaucoma

By potentiating the effects of the natriuretic peptides, compounds ofthe invention are expected to be useful to treat glaucoma. See, forexample, Diestelhorst et al. (1989) International Ophthalmology12:99-101. When used to treat glaucoma, compounds of the invention maybe combined with one or more additional anti-glaucoma agents.

Pain Relief

As NEP inhibitors, compounds of the invention are expected to inhibitthe degradation of endogenous enkephalins and thus such compounds mayalso find utility as analgesics. See, for example, Roques et al. (1980)Nature 288:286-288 and Thanawala et al. (2008) Current Drug Targets9:887-894. When used to treat pain, compounds of the invention may becombined with one or more additional antinociceptive drugs such asaminopeptidase N or dipeptidyl peptidase III inhibitors, non-steroidalanti-inflammatory agents, monoamine reuptake inhibitors, musclerelaxants, NMDA receptor antagonists, opioid receptor agonists,5-HT_(1D) serotonin receptor agonists, and tricyclic antidepressants.

Other Utilities

Due to their NEP inhibition properties, compounds of the invention arealso expected to be useful as antitussive agents, as well as findutility in the treatment of portal hypertension associated with livercirrhosis (see Sansoe et al. (2005) J. Hepatol. 43:791-798), cancer (seeVesely (2005) J. Investigative Med. 53:360-365), depression (see Nobleet al. (2007) Exp. Opin. Ther. Targets 11:145-159), menstrual disorders,preterm labor, pre-eclampsia, endometriosis, reproductive disorders (forexample, male and female infertility, polycystic ovarian syndrome,implantation failure), and male and female sexual dysfunction, includingmale erectile dysfunction and female sexual arousal disorder. Morespecifically, the compounds of the invention are expected to be usefulin treating female sexual dysfunction (see Pryde et al. (2006) J. Med.Chem. 49:4409-4424), which is often defined as a female patient'sdifficulty or inability to find satisfaction in sexual expression. Thiscovers a variety of diverse female sexual disorders including, by way ofillustration and not limitation, hypoactive sexual desire disorder,sexual arousal disorder, orgasmic disorder and sexual pain disorder.When used to treat such disorders, especially female sexual dysfunction,compounds of the invention may be combined with one or more of thefollowing secondary agents: PDE-V inhibitors, dopamine agonists,estrogen receptor agonists and/or antagonists, androgens, and estrogens.Due to their NEP inhibition properties, compounds of the invention arealso expected to have anti-inflammatory properties, and are expected tohave utility as such, particularly when used in combination withstatins.

Recent studies suggest that NEP plays a role in regulating nervefunction in insulin-deficient diabetes and diet induced obesity. Coppeyet al. (2011) Neuropharmacology 60:259-266. Therefore, due to their NEPinhibition properties, compounds of the invention are also expected tobe useful in providing protection from nerve impairment caused bydiabetes or diet induced obesity.

The amount of the compound of the invention administered per dose or thetotal amount administered per day may be predetermined or it may bedetermined on an individual patient basis by taking into considerationnumerous factors, including the nature and severity of the patient'scondition, the condition being treated, the age, weight, and generalhealth of the patient, the tolerance of the patient to the active agent,the route of administration, pharmacological considerations such as theactivity, efficacy, pharmacokinetics and toxicology profiles of thecompound and any secondary agents being administered, and the like.Treatment of a patient suffering from a disease or medical condition(such as hypertension) can begin with a predetermined dosage or a dosagedetermined by the treating physician, and will continue for a period oftime necessary to prevent, ameliorate, suppress, or alleviate thesymptoms of the disease or medical condition. Patients undergoing suchtreatment will typically be monitored on a routine basis to determinethe effectiveness of therapy. For example, in treating hypertension,blood pressure measurements may be used to determine the effectivenessof treatment. Similar indicators for other diseases and conditionsdescribed herein, are well known and are readily available to thetreating physician. Continuous monitoring by the physician will insurethat the optimal amount of the compound of the invention will beadministered at any given time, as well as facilitating thedetermination of the duration of treatment. This is of particular valuewhen secondary agents are also being administered, as their selection,dosage, and duration of therapy may also require adjustment. In thisway, the treatment regimen and dosing schedule can be adjusted over thecourse of therapy so that the lowest amount of active agent thatexhibits the desired effectiveness is administered and, further, thatadministration is continued only so long as is necessary to successfullytreat the disease or medical condition.

Research Tools

Since the compounds of the invention are metabolized in vivo tocompounds having activity as neprilysin inhibitors, they are also usefulas a research tools for investigating or studying biological systems orsamples having a NEP enzyme, for example to study diseases where the NEPenzyme or its peptide substrates plays a role. Accordingly, one aspectof the invention relates to a method of using a compound of theinvention as a research tool, comprising conducting a biological assayusing a compound of the invention. Any suitable biological system orsample having a NEP enzyme may be employed in such studies which may beconducted either in vitro or in vivo. Representative biological systemsor samples suitable for such studies include, but are not limited to,cells, cellular extracts, plasma membranes, tissue samples, isolatedorgans, mammals (such as mice, rats, guinea pigs, rabbits, dogs, pigs,humans, and so forth), and the like, with mammals being of particularinterest. In one particular embodiment of the invention, NEP enzymeactivity in a mammal is inhibited by administering a NEP-inhibitingamount of a compound of the invention. These compounds can also be usedas research tools by conducting biological assays using such compounds.

When used as a research tool, a biological system or sample comprising aNEP enzyme is typically contacted with a NEP enzyme-inhibiting amount ofa compound of the invention. After the biological system or sample isexposed to the compound, the effects of inhibiting the NEP enzyme aredetermined using conventional procedures and equipment, such as bymeasuring receptor binding in a binding assay or measuringligand-mediated changes in a functional assay. Exposure encompassescontacting cells or tissue with the compound, administering thecrystalline compound to a mammal, for example by i.p., p.o, i.v., s.c.,or inhaled administration, and so forth. This determining step caninvolve measuring a response (a quantitative analysis) or can involvemaking an observation (a qualitative analysis). Measuring a responseinvolves, for example, determining the effects of the compound on thebiological system or sample using conventional procedures and equipment,such as enzyme activity assays and measuring enzyme substrate or productmediated changes in functional assays. The assay results can be used todetermine the activity level as well as the amount of compound necessaryto achieve the desired result, that is, a NEP enzyme-inhibiting amount.Typically, the determining step will involve determining the effects ofinhibiting the NEP enzyme.

Additionally, the compounds of the invention can be used as researchtools for evaluating other chemical compounds, and thus are also usefulin screening assays to discover, for example, new compounds havingNEP-inhibiting activity. Thus another aspect of the invention relates toa method of evaluating a test compound in a biological assay,comprising: (a) conducting a biological assay with a test compound toprovide a first assay value; (b) conducting the biological assay with acompound of the invention to provide a second assay value; wherein step(a) is conducted either before, after or concurrently with step (b); and(c) comparing the first assay value from step (a) with the second assayvalue from step (b). Exemplary biological assays include a NEP enzymeinhibition assay. In this manner, the compounds of the invention areused as standards in an assay to allow comparison of the resultsobtained with a test compound and with the compound of the invention toidentify those test compounds that have about equal or superioractivity, if any. For example, pK_(i) data for a test compound or agroup of test compounds is compared to the pK_(i) data for a compound ofthe invention to identify those test compounds that have the desiredproperties, for example, test compounds having a pK_(i) value aboutequal or superior to the compound of the invention, if any. This aspectof the invention includes, as separate embodiments, both the generationof comparison data (using the appropriate assays) and the analysis oftest data to identify test compounds of interest.

Still another aspect of the invention relates to a method of studying abiological system or sample comprising a NEP enzyme, the methodcomprising: (a) contacting the biological system or sample with acompound of the invention; and (b) determining the effects caused by thecompound on the biological system or sample.

Pharmaceutical Compositions and Formulations

Compounds of the invention are typically administered to a patient inthe form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to, oral,rectal, vaginal, nasal, inhaled, topical (including transdermal),ocular, and parenteral modes of administration. Further, the compoundsof the invention may be administered, for example orally, in multipledoses per day (for example, two, three, or four times daily), in asingle daily dose or a single weekly dose. It will be understood thatany form of the compounds of the invention, (that is, free base, freeacid, pharmaceutically acceptable salt, solvate, etc.) that is suitablefor the particular mode of administration can be used in thepharmaceutical compositions discussed herein.

Accordingly, in one embodiment, the invention relates to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of the invention. The compositions may containother therapeutic and/or formulating agents if desired. When discussingcompositions, the “compound of the invention” may also be referred toherein as the “active agent,” to distinguish it from other components ofthe formulation, such as the carrier. Thus, it is understood that theterm “active agent” includes compounds of formula I as well aspharmaceutically acceptable salts, solvates and prodrugs of thatcompound.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the invention. Thoseskilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,such as in bulk compositions, or less than a therapeutically effectiveamount, that is, individual unit doses designed for multipleadministration to achieve a therapeutically effective amount. Typically,the composition will contain from about 0.01-95 wt % of active agent,including, from about 0.01-30 wt %, such as from about 0.01-10 wt %,with the actual amount depending upon the formulation itself, the routeof administration, the frequency of dosing, and so forth. In oneembodiment, a composition suitable for an oral dosage form, for example,may contain about 5-70 wt %, or from about 10-60 wt % of active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable composition for a particular mode of administration iswell within the scope of those skilled in the pharmaceutical arts.Additionally, carriers or excipients used in such compositions arecommercially available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; compressed propellant gases, such aschlorofluorocarbons and hydrofluorocarbons; and other non-toxiccompatible substances employed in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with a pharmaceuticallyacceptable carrier and one or more optional ingredients. The resultinguniformly blended mixture may then be shaped or loaded into tablets,capsules, pills, canisters, cartridges, dispensers and the like usingconventional procedures and equipment.

In one embodiment, the pharmaceutical compositions are suitable for oraladministration. Suitable compositions for oral administration may be inthe form of capsules, tablets, pills, lozenges, cachets, dragees,powders, granules; solutions or suspensions in an aqueous or non-aqueousliquid; oil-in-water or water-in-oil liquid emulsions; elixirs orsyrups; and the like; each containing a predetermined amount of theactive agent.

When intended for oral administration in a solid dosage form (capsules,tablets, pills and the like), the composition will typically comprisethe active agent and one or more pharmaceutically acceptable carriers,such as sodium citrate or dicalcium phosphate. Solid dosage forms mayalso comprise: fillers or extenders, such as starches, microcrystallinecellulose, lactose, sucrose, glucose, mannitol, and/or silicic acid;binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; humectants, such as glycerol;disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; solution retarding agents, such as paraffin; absorptionaccelerators, such as quaternary ammonium compounds; wetting agents,such as cetyl alcohol and/or glycerol monostearate; absorbents, such askaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants may also be presentin the pharmaceutical compositions. Exemplary coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate,carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, and the like. Examples of pharmaceutically acceptableantioxidants include: water-soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfate sodiumsulfite and the like; oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin,propyl gallate, alpha-tocopherol, and the like; and metal-chelatingagents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions may also be formulated to provide slow or controlledrelease of the active agent using, by way of example, hydroxypropylmethyl cellulose in varying proportions or other polymer matrices,liposomes and/or microspheres. In addition, the pharmaceuticalcompositions of the invention may contain opacifying agents and may beformulated so that they release the active agent only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. The activeagent can also be in micro-encapsulated form, optionally with one ormore of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (for example, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.Suspensions may contain suspending agents such as, for example,ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminium metahydroxide, bentonite,agar-agar and tragacanth, and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof the invention may be packaged in a unit dosage form. The term “unitdosage form” refers to a physically discrete unit suitable for dosing apatient, that is, each unit containing a predetermined quantity of theactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

In another embodiment, the compositions of the invention are suitablefor inhaled administration, and will typically be in the form of anaerosol or a powder. Such compositions are generally administered usingwell-known delivery devices, such as a nebulizer, dry powder, ormetered-dose inhaler. Nebulizer devices produce a stream of highvelocity air that causes the composition to spray as a mist that iscarried into a patient's respiratory tract. An exemplary nebulizerformulation comprises the active agent dissolved in a carrier to form asolution, or micronized and combined with a carrier to form a suspensionof micronized particles of respirable size. Dry powder inhalersadminister the active agent as a free-flowing powder that is dispersedin a patient's air-stream during inspiration. An exemplary dry powderformulation comprises the active agent dry-blended with an excipientsuch as lactose, starch, mannitol, dextrose, polylactic acid,polylactide-co-glycolide, and combinations thereof. Metered-doseinhalers discharge a measured amount of the active agent usingcompressed propellant gas. An exemplary metered-dose formulationcomprises a solution or suspension of the active agent in a liquefiedpropellant, such as a chlorofluorocarbon or hydrofluoroalkane. Optionalcomponents of such formulations include co-solvents, such as ethanol orpentane, and surfactants, such as sorbitan trioleate, oleic acid,lecithin, glycerin, and sodium lauryl sulfate. Such compositions aretypically prepared by adding chilled or pressurized hydrofluoroalkane toa suitable container containing the active agent, ethanol (if present)and the surfactant (if present). To prepare a suspension, the activeagent is micronized and then combined with the propellant.Alternatively, a suspension formulation can be prepared by spray dryinga coating of surfactant on micronized particles of the active agent. Theformulation is then loaded into an aerosol canister, which forms aportion of the inhaler.

Compounds of the invention can also be administered parenterally (forexample, by subcutaneous, intravenous, intramuscular, or intraperitonealinjection). For such administration, the active agent is provided in asterile solution, suspension, or emulsion. Exemplary solvents forpreparing such formulations include water, saline, low molecular weightalcohols such as propylene glycol, polyethylene glycol, oils, gelatin,fatty acid esters such as ethyl oleate, and the like. Parenteralformulations may also contain one or more anti-oxidants, solubilizers,stabilizers, preservatives, wetting agents, emulsifiers, and dispersingagents. Surfactants, additional stabilizing agents or pH-adjustingagents (acids, bases or buffers) and anti-oxidants are particularlyuseful to provide stability to the formulation, for example, to minimizeor avoid hydrolysis of ester and amide linkages, or dimerization ofthiols that may be present in the compound. These formulations may berendered sterile by use of a sterile injectable medium, a sterilizingagent, filtration, irradiation, or heat. In one particular embodiment,the parenteral formulation comprises an aqueous cyclodextrin solution asthe pharmaceutically acceptable carrier. Suitable cyclodextrins includecyclic molecules containing six or more α-D-glucopyranose units linkedat the 1,4 positions by a linkages as in amylase, β-cyclodextrin orcycloheptaamylose. Exemplary cyclodextrins include cyclodextrinderivatives such as hydroxypropyl and sulfobutyl ether cyclodextrinssuch as hydroxypropyl-β-cyclodextrin and sulfobutyl etherβ-cyclodextrin. Exemplary buffers for such formulations includecarboxylic acid-based buffers such as citrate, lactate and maleatebuffer solutions.

Compounds of the invention can also be administered transdermally usingknown transdermal delivery systems and excipients. For example, thecompound can be admixed with permeation enhancers, such as propyleneglycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and thelike, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

Secondary Agents

The compounds of the invention may be useful as the sole treatment of adisease or may be combined with one or more additional therapeuticagents in order to obtain the desired therapeutic effect. Thus, in oneembodiment, pharmaceutical compositions of the invention contain otherdrugs that are co-administered with a compound of the invention. Forexample, the composition may further comprise one or more drugs (alsoreferred to as “secondary agents(s)”). Such therapeutic agents are wellknown in the art, and include adenosine receptor antagonists,α-adrenergic receptor antagonists, β₁-adrenergic receptor antagonists,β₂-adrenergic receptor agonists, dual-acting β-adrenergic receptorantagonist/α₁-receptor antagonists, advanced glycation end productbreakers, aldosterone antagonists, aldosterone synthase inhibitors,aminopeptidase N inhibitors, androgens, angiotensin-converting enzymeinhibitors and dual-acting angiotensin-converting enzyme/neprilysininhibitors, angiotensin-converting enzyme 2 activators and stimulators,angiotensin-II vaccines, anticoagulants, anti-diabetic agents,antidiarrheal agents, anti-glaucoma agents, anti-lipid agents,antinociceptive agents, anti-thrombotic agents, AT₁ receptor antagonistsand dual-acting AT₁ receptor antagonist/neprilysin inhibitors andmultifunctional angiotensin receptor blockers, bradykinin receptorantagonists, calcium channel blockers, chymase inhibitors, digoxin,diuretics, dopamine agonists, endothelin converting enzyme inhibitors,endothelin receptor antagonists, HMG-CoA reductase inhibitors,estrogens, estrogen receptor agonists and/or antagonists, monoaminereuptake inhibitors, muscle relaxants, natriuretic peptides and theiranalogs, natriuretic peptide clearance receptor antagonists, neprilysininhibitors, nitric oxide donors, non-steroidal anti-inflammatory agents,N-methyl d-aspartate receptor antagonists, opioid receptor agonists,phosphodiesterase inhibitors, prostaglandin analogs, prostaglandinreceptor agonists, renin inhibitors, selective serotonin reuptakeinhibitors, sodium channel blocker, soluble guanylate cyclasestimulators and activators, tricyclic antidepressants, vasopressinreceptor antagonists, and combinations thereof. Specific examples ofthese agents are detailed herein.

Accordingly, in yet another aspect of the invention, a pharmaceuticalcomposition comprises a compound of the invention, a second activeagent, and a pharmaceutically acceptable carrier. Third, fourth etc.active agents may also be included in the composition. In combinationtherapy, the amount of compound of the invention that is administered,as well as the amount of secondary agents, may be less than the amounttypically administered in monotherapy.

Compounds of the invention may be physically mixed with the secondactive agent to form a composition containing both agents; or each agentmay be present in separate and distinct compositions which areadministered to the patient simultaneously or at separate times. Forexample, a compound of the invention can be combined with a secondactive agent using conventional procedures and equipment to form acombination of active agents comprising a compound of the invention anda second active agent. Additionally, the active agents may be combinedwith a pharmaceutically acceptable carrier to form a pharmaceuticalcomposition comprising a compound of the invention, a second activeagent and a pharmaceutically acceptable carrier. In this embodiment, thecomponents of the composition are typically mixed or blended to create aphysical mixture. The physical mixture is then administered in atherapeutically effective amount using any of the routes describedherein.

Alternatively, the active agents may remain separate and distinct beforeadministration to the patient. In this embodiment, the agents are notphysically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together in akit. When administered at separate times, the secondary agent willtypically be administered less than 24 hours after administration of thecompound of the invention, ranging anywhere from concurrent withadministration of the compound of the invention to about 24 hourspost-dose. This is also referred to as sequential administration. Thus,a compound of the invention can be orally administered simultaneously orsequentially with another active agent using two tablets, with onetablet for each active agent, where sequential may mean beingadministered immediately after administration of the compound of theinvention or at some predetermined time later (for example, one hourlater or three hours later). It is also contemplated that the secondaryagent may be administered more than 24 hours after administration of thecompound of the invention. Alternatively, the combination may beadministered by different routes of administration, that is, one orallyand the other by inhalation.

In one embodiment, the kit comprises a first dosage form comprising acompound of the invention and at least one additional dosage formcomprising one or more of the secondary agents set forth herein, inquantities sufficient to carry out the methods of the invention. Thefirst dosage form and the second (or third, etc.) dosage form togethercomprise a therapeutically effective amount of active agents for thetreatment or prevention of a disease or medical condition in a patient.

Secondary agent(s), when included, are present in a therapeuticallyeffective amount such that they are typically administered in an amountthat produces a therapeutically beneficial effect when co-administeredwith a compound of the invention. The secondary agent can be in the formof a pharmaceutically acceptable salt, solvate, optically purestereoisomer, and so forth. The secondary agent may also be in the formof a prodrug, for example, a compound having a carboxylic acid groupthat has been esterified. Thus, secondary agents listed herein areintended to include all such forms, and are commercially available orcan be prepared using conventional procedures and reagents.

In one embodiment, compounds of the invention are administered incombination with an adenosine receptor antagonist, representativeexamples of which include, but are not limited to, naxifylline,rolofylline, SLV-320, theophylline, and tonapofylline.

In one embodiment, compounds of the invention are administered incombination with an α-adrenergic receptor antagonist, representativeexamples of which include, but are not limited to, doxazosin, prazosin,tamsulosin, and terazosin.

Compounds of the invention may also be administered in combination witha β₁-adrenergic receptor antagonist (“β₁-blockers”). Representativeβ₁-blockers include, but are not limited to, acebutolol, alprenolol,amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol,bisoprolol, bopindolol, bucindolol, bucumolol, bufetolol, bufuralol,bunitrolol, bupranolol, bubridine, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol,esmolol, indenolol, labetolol, levobunolol, mepindolol, metipranolol,metoprolol such as metoprolol succinate and metoprolol tartrate,moprolol, nadolol, nadoxolol, nebivalol, nipradilol, oxprenolol,penbutolol, perbutolol, pindolol, practolol, pronethalol, propranolol,sotalol, sufinalol, talindol, tertatolol, tilisolol, timolol,toliprolol, xibenolol, and combinations thereof. In one particularembodiment, the β₁-antagonist is selected from atenolol, bisoprolol,metoprolol, propranolol, sotalol, and combinations thereof. Typically,the β₁-blocker will be administered in an amount sufficient to providefrom about 2-900 mg per dose.

In one embodiment, compounds of the invention are administered incombination with a β₂-adrenergic receptor agonist, representativeexamples of which include, but are not limited to, albuterol,bitolterol, fenoterol, formoterol, indacaterol, isoetharine,levalbuterol, metaproterenol, pirbuterol, salbutamol, salmefamol,salmeterol, terbutaline, vilanterol, and the like Typically, theβ₂-adrenergic receptor agonist will be administered in an amountsufficient to provide from about 0.05-500 μg per dose.

In one embodiment, compounds of the invention are administered incombination with an advanced glycation end product (AGE) breaker,examples of which include, by way of illustration and not limitation,alagebrium (or ALT-711), and TRC4149.

In another embodiment, compounds of the invention are administered incombination with an aldosterone antagonist, representative examples ofwhich include, but are not limited to, eplerenone, spironolactone, andcombinations thereof. Typically, the aldosterone antagonist will beadministered in an amount sufficient to provide from about 5-300 mg perday.

In one embodiment, compounds of the invention are administered incombination with an aminopeptidase N or dipeptidyl peptidase IIIinhibitor, examples of which include, by way of illustration and notlimitation, bestatin and PC18 (2-amino-4-methylsulfonyl butane thiol,methionine thiol).

Compounds of the invention can also be administered in combination withan angiotensin-converting enzyme (ACE) inhibitor. Representative ACEinhibitors include, but are not limited to, accupril, alacepril,benazepril, benazeprilat, captopril, ceranapril, cilazapril, delapril,enalapril, enalaprilat, fosinopril, fosinoprilat, imidapril, lisinopril,moexipril, monopril, moveltipril, pentopril, perindopril, quinapril,quinaprilat, ramipril, ramiprilat, saralasin acetate, spirapril,temocapril, trandolapril, zofenopril, and combinations thereof.

In a particular embodiment, the ACE inhibitor is selected from:benazepril, captopril, enalapril, lisinopril, ramipril, and combinationsthereof. Typically, the ACE inhibitor will be administered in an amountsufficient to provide from about 1-150 mg per day. In anotherembodiment, compounds of the invention are administered in combinationwith a dual-acting angiotensin-converting enzyme/neprilysin (ACE/NEP)inhibitor, examples of which include, but are not limited to: AVE-0848((4S,7S,12bR)-7-[3-methyl-2(S)-sulfanylbutyramido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]-benzazepine-4-carboxylicacid); AVE-7688 (ilepatril) and its parent compound; BMS-182657(2-[2-oxo-3(S)-[3-phenyl-2(S)-sulfanylpropionamido]-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]aceticacid); CGS-35601(N-[1-[4-methyl-2(S)-sulfanylpentanamido]cyclopentyl-carbonyl]-L-tryptophan);fasidotril; fasidotrilate; enalaprilat; ER-32935((3R,6S,9aR)-6-[3(S)-methyl-2(S)-sulfanylpentanamido]-5-oxoperhydrothiazolo[3,2-a]azepine-3-carboxylicacid); gempatrilat; MDL-101264((4S,7S,12bR)-7-[2(S)-(2-morpholinoacetylthio)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); MDL-101287 ([4S-[4α,7α(R*),12bβ]]-7-[2-(carboxymethyl)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); omapatrilat; RB-105(N-[2(S)-(mercaptomethyl)-3(R)-phenylbutyl]-L-alanine); sampatrilat;SA-898((2R,4R)—N-[2-(2-hydroxyphenyl)-3-(3-mercaptopropionyl)thiazolidin-4-ylcarbonyl]-L-phenylalanine);Sch-50690(N-[1(S)-carboxy-2-[N2-(methanesulfonyl)-L-lysylamino]ethyl]-L-valyl-L-tyrosine);and combinations thereof, may also be included. In one particularembodiment, the ACE/NEP inhibitor is selected from: AVE-7688,enalaprilat, fasidotril, fasidotrilate, omapatrilat, sampatrilat, andcombinations thereof.

In one embodiment, compounds of the invention are administered incombination with an angiotensin-converting enzyme 2 (ACE2) activator orstimulator.

In one embodiment, compounds of the invention are administered incombination with an angiotensin-II vaccine, examples of which include,but are not limited to ATR12181 and CYT006-AngQb.

In one embodiment, compounds of the invention are administered incombination with an anticoagulant, representative examples of whichinclude, but are not limited to: coumarins such as warfarin; heparin;and direct thrombin inhibitors such as argatroban, bivalirudin,dabigatran, and lepirudin.

In yet another embodiment, compounds of the invention are administeredin combination with an anti-diabetic agent. Representative anti-diabeticagents include injectable drugs as well as orally effective drugs, andcombinations thereof. Examples of injectable drugs include, but are notlimited to, insulin and insulin derivatives. Examples of orallyeffective drugs include, but are not limited to: biguanides such asmetformin; glucagon antagonists; α-glucosidase inhibitors such asacarbose and miglitol; dipeptidyl peptidase IV inhibitors (DPP-IVinhibitors) such as alogliptin, denagliptin, linagliptin, saxagliptin,sitagliptin, and vildagliptin; meglitinides such as repaglinide;oxadiazolidinediones; sulfonylureas such as chlorpropamide, glimepiride,glipizide, glyburide, and tolazamide; thiazolidinediones such aspioglitazone and rosiglitazone; and combinations thereof.

In another embodiment, compounds of the invention are administered incombination with antidiarrheal treatments. Representative treatmentoptions include, but are not limited to, oral rehydration solutions(ORS), loperamide, diphenoxylate, and bismuth subsalicylate.

In yet another embodiment, a compound of the invention is administeredin combination with an anti-glaucoma agent. Representative anti-glaucomaagents include, but are not limited to: α-adrenergic agonists such asbrimonidine; β₁-adrenergic receptor antagonists; topical β₁-blockerssuch as betaxolol, levobunolol, and timolol; carbonic anhydraseinhibitors such as acetazolamide, brinzolamide, or dorzolamide;cholinergic agonists such as cevimeline and DMXB-anabaseine; epinephrinecompounds; miotics such as pilocarpine; and prostaglandin analogs.

In yet another embodiment, compounds of the invention are administeredin combination with an anti-lipid agent. Representative anti-lipidagents include, but are not limited to: cholesteryl ester transferprotein inhibitors (CETPs) such as anacetrapib, dalcetrapib, andtorcetrapib; statins such as atorvastatin, fluvastatin, lovastatin,pravastatin, rosuvastatin and simvastatin; and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with an anti-thrombotic agent. Representativeanti-thrombotic agents include, but are not limited to: aspirin;anti-platelet agents such as clopidogrel, prasugrel, and ticlopidine;heparin, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with an AT₁ receptor antagonist, also known as angiotensinII type 1 receptor blockers (ARBs). Representative ARBs include, but arenot limited to, abitesartan, azilsartan (e.g., azilsartan medoxomil),benzyllosartan, candesartan, candesartan cilexetil, elisartan,embusartan, enoltasosartan, eprosartan, EXP3174, fonsartan, forasartan,glycyllosartan, irbesartan, isoteoline, losartan, medoxomil,milfasartan, olmesartan (e.g., olmesartan medoxomil), opomisartan,pratosartan, ripisartan, saprisartan, saralasin, sarmesin, TAK-591,tasosartan, telmisartan, valsartan, zolasartan, and combinationsthereof. In a particular embodiment, the ARB is selected from azilsartanmedoxomil, candesartan cilexetil, eprosartan, irbesartan, losartan,olmesartan medoxomil, saprisartan, tasosartan, telmisartan, valsartan,and combinations thereof. Exemplary salts and/or prodrugs includecandesartan cilexetil, eprosartan mesylate, losartan potassium salt, andolmesartan medoxomil. Typically, the ARB will be administered in anamount sufficient to provide from about 4-600 mg per dose, withexemplary daily dosages ranging from 20-320 mg per day.

Compounds of the invention may also be administered in combination witha dual-acting agent, such as an AT₁ receptor antagonist/neprilysininhibitor (ARB/NEP) inhibitor, examples of which include, but are notlimited to, compounds described in U.S. Publication Nos. 2008/0269305and 2009/0023228, both to Allegretti et al. filed on Apr. 23, 2008, suchas the compound,4′-{2-ethoxy-4-ethyl-5-[((S)-2-mercapto-4-methylpentanoylamino)-methyl]imidazol-1-ylmethyl}-3′-fluorobiphenyl-2-carboxylicacid.

Compounds of the invention may also be administered in combination withmultifunctional angiotensin receptor blockers as described in Kurtz &Klein (2009) Hypertension Research 32:826-834.

In one embodiment, compounds of the invention are administered incombination with a bradykinin receptor antagonist, for example,icatibant (HOE-140). It is expected that this combination therapy maypresent the advantage of preventing angioedema or other unwantedconsequences of elevated bradykinin levels.

In one embodiment, compounds of the invention are administered incombination with a calcium channel blocker. Representative calciumchannel blockers include, but are not limited to, amlodipine, anipamil,aranipine, bamidipine, bencyclane, benidipine, bepridil, clentiazem,cilnidipine, cinnarizine, diltiazem, efonidipine, elgodipine, etafenone,felodipine, fendiline, flunarizine, gallopamil, isradipine, lacidipine,lercanidipine, lidoflazine, lomerizine, manidipine, mibefradil,nicardipine, nifedipine, niguldipine, niludipine, nilvadipine,nimodipine, nisoldipine, nitrendipine, nivaldipine, perhexiline,prenylamine, ryosidine, semotiadil, terodiline, tiapamil, verapamil, andcombinations thereof. In a particular embodiment, the calcium channelblocker is selected from amlodipine, bepridil, diltiazem, felodipine,isradipine, lacidipine, nicardipine, nifedipine, niguldipine,niludipine, nimodipine, nisoldipine, ryosidine, verapamil, andcombinations thereof. Typically, the calcium channel blocker will beadministered in an amount sufficient to provide from about 2-500 mg perdose.

In one embodiment, compounds of the invention are administered incombination with a chymase inhibitor, such as TPC-806 and2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[{3,4-dioxo-1-phenyl-7-(2-pyridyloxy)}-2-heptyl]acetamide(NK3201).

In one embodiment, compounds of the invention are administered incombination with a diuretic. Representative diuretics include, but arenot limited to: carbonic anhydrase inhibitors such as acetazolamide anddichlorphenamide; loop diuretics, which include sulfonamide derivativessuch as acetazolamide, ambuside, azosemide, bumetanide, butazolamide,chloraminophenamide, clofenamide, clopamide, clorexolone, disulfamide,ethoxzolamide, furosemide, mefruside, methazolamide, piretanide,torsemide, tripamide, and xipamide, as well as non-sulfonamide diureticssuch as ethacrynic acid and other phenoxyacetic acid compounds such astienilic acid, indacrinone and quincarbate; osmotic diuretics such asmannitol; potassium-sparing diuretics, which include aldosteroneantagonists such as spironolactone, and Na⁺ channel inhibitors such asamiloride and triamterene; thiazide and thiazide-like diuretics such asalthiazide, bendroflumethiazide, benzylhydrochlorothiazide,benzthiazide, buthiazide, chlorthalidone, chlorothiazide,cyclopenthiazide, cyclothiazide, epithiazide, ethiazide, fenquizone,flumethiazide, hydrochlorothiazide, hydroflumethiazide, indapamide,methylclothiazide, meticrane, metolazone, paraflutizide, polythiazide,quinethazone, teclothiazide, and trichloromethiazide; and combinationsthereof. In a particular embodiment, the diuretic is selected fromamiloride, bumetanide, chlorothiazide, chlorthalidone, dichlorphenamide,ethacrynic acid, furosemide, hydrochlorothiazide, hydroflumethiazide,indapamide, methylclothiazide, metolazone, torsemide, triamterene, andcombinations thereof. The diuretic will be administered in an amountsufficient to provide from about 5-50 mg per day, more typically 6-25 mgper day, with common dosages being 6.25 mg, 12.5 mg or 25 mg per day.

Compounds of the invention may also be administered in combination withan endothelin converting enzyme (ECE) inhibitor, examples of whichinclude, but are not limited to, phosphoramidon, CGS 26303, andcombinations thereof.

In a particular embodiment, compounds of the invention are administeredin combination with an endothelin receptor antagonist. Representativeendothelin receptor antagonists include, but are not limited to:selective endothelin receptor antagonists that affect endothelin Areceptors, such as avosentan, ambrisentan, atrasentan, BQ-123,clazosentan, darusentan, sitaxentan, and zibotentan; and dual endothelinreceptor antagonists that affect both endothelin A and B receptors, suchas bosentan, macitentan, tezosentan).

In yet another embodiment, a compound of the invention is administeredin combination with one or more HMG-CoA reductase inhibitors, which arealso known as statins. Representative statins include, but are notlimited to, atorvastatin, fluvastatin, lovastatin, pitavastatin,pravastatin, rosuvastatin and simvastatin.

In one embodiment, compounds of the invention are administered incombination with a monoamine reuptake inhibitor, examples of whichinclude, by way of illustration and not limitation, norepinephrinereuptake inhibitors such as atomoxetine, buproprion and the buproprionmetabolite hydroxybuproprion, maprotiline, reboxetine, and viloxazine;selective serotonin reuptake inhibitors (SSRIs) such as citalopram andthe citalopram metabolite desmethylcitalopram, dapoxetine, escitalopram(e.g., escitalopram oxalate), fluoxetine and the fluoxetine desmethylmetabolite norfluoxetine, fluvoxamine (e.g., fluvoxamine maleate),paroxetine, sertraline and the sertraline metabolite demethylsertraline;dual serotonin-norepinephrine reuptake inhibitors (SNRIs) such asbicifadine, duloxetine, milnacipran, nefazodone, and venlafaxine; andcombinations thereof.

In another embodiment, compounds of the invention are administered incombination with a muscle relaxant, examples of which include, but arenot limited to: carisoprodol, chlorzoxazone, cyclobenzaprine,diflunisal, metaxalone, methocarbamol, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a natriuretic peptide or analog, examples of whichinclude but are not limited to: carperitide, CD-NP (Nile Therapeutics),CU-NP, nesiritide, PL-3994 (Palatin Technologies, Inc.), ularitide,cenderitide, and compounds described in Ogawa et al (2004) J. Biol.Chem. 279:28625-31. These compounds are also referred to as natriureticpeptide receptor-A (NPR-A) agonists. In another embodiment, compounds ofthe invention are administered in combination with a natriuretic peptideclearance receptor (NPR-C) antagonist such as SC-46542, cANF (4-23), andAP-811 (Veale (2000) Bioorg Med Chem Lett 10:1949-52). For example,AP-811 has shown synergy when combined with the NEP inhibitor, thiorphan(Wegner (1995) Clin. Exper. Hypert. 17:861-876).

In another embodiment, compounds of the invention are administered incombination with a neprilysin (NEP) inhibitor. Representative NEPinhibitors include, but are not limited to: AHU-377; candoxatril;candoxatrilat; dexecadotril((+)-N-[2(R)-(acetylthiomethyl)-3-phenylpropionyl]glycine benzyl ester);CGS-24128(3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-24592((S)-3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-25155(N-[9(R)-(acetylthiomethyl)-10-oxo-1-azacyclodecan-2(S)-ylcarbonyl]-4(R)-hydroxy-L-prolinebenzyl ester); 3-(1-carbamoylcyclohexyl)propionic acid derivativesdescribed in WO 2006/027680 to Hepworth et al. (Pfizer Inc.); JMV-390-1(2(R)-benzyl-3-(N-hydroxycarbamoyl)propionyl-L-isoleucyl-L-leucine);ecadotril; phosphoramidon; retrothiorphan; RU-42827(2-(mercaptomethyl)-N-(4-pyridinyl)benzenepropionamide); RU-44004(N-(4-morpholinyl)-3-phenyl-2-(sulfanylmethyl)propionamide); SCH-32615((S)—N—[N-(1-carboxy-2-phenylethyl)-L-phenylalanyl]-β-alanine) and itsprodrug SCH-34826((S)—N—[N-[1-[[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-β-alanine);sialorphin; SCH-42495(N-[2(S)-(acetylsulfanylmethyl)-3-(2-methylphenyl)propionyl]-L-methionineethyl ester); spinorphin; SQ-28132(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]leucine); SQ-28603(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-β-alanine); SQ-29072(7-[[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]amino]heptanoic acid);thiorphan and its prodrug racecadotril; UK-69578(cis-4-[[[1-[2-carboxy-3-(2-methoxyethoxy)propyl]cyclopentyl]carbonyl]amino]cyclohexanecarboxylicacid); UK-447,841(2-{1-[3-(4-chlorophenyl)propylcarbamoyl]-cyclopentylmethyl}-4-methoxybutyricacid); UK-505,749((R)-2-methyl-3-{1-[3-(2-methylbenzothiazol-6-yl)propylcarbamoyl]cyclopentyl}propionicacid); 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid and 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid ethyl ester (WO 2007/056546); daglutril[(3S,2′R)-3-{1-[2′-(ethoxycarbonyl)-4′-phenylbutyl]-cyclopentan-1-carbonylamino}-2,3,4,5-tetrahydro-2-oxo-1H-1-benzazepine-1-aceticacid] described in WO 2007/106708 to Khder et al. (Novartis AG); andcombinations thereof. In a particular embodiment, the NEP inhibitor isselected from AHU-377, candoxatril, candoxatrilat, CGS-24128,phosphoramidon, SCH-32615, SCH-34826, SQ-28603, thiorphan, andcombinations thereof. In a particular embodiment, the NEP inhibitor is acompound such as daglutril or CGS-26303([N-[2-(biphenyl-4-yl)-1(S)-(1H-tetrazol-5-yl)ethyl]amino]methylphosphonicacid), which have activity both as inhibitors of the endothelinconverting enzyme (ECE) and of NEP. Other dual acting ECE/NEP compoundscan also be used. The NEP inhibitor will be administered in an amountsufficient to provide from about 20-800 mg per day, with typical dailydosages ranging from 50-700 mg per day, more commonly 100-600 or 100-300mg per day.

In one embodiment, compounds of the invention are administered incombination with a nitric oxide donor, examples of which include, butare not limited to nicorandil; organic nitrates such as pentaerythritoltetranitrate; and sydnonimines such as linsidomine and molsidomine.

In yet another embodiment, compounds of the invention are administeredin combination with a non-steroidal anti-inflammatory agent (NSAID).Representative NSAIDs include, but are not limited to: acemetacin,acetyl salicylic acid, alclofenac, alminoprofen, amfenac, amiprilose,aloxiprin, anirolac, apazone, azapropazone, benorilate, benoxaprofen,bezpiperylon, broperamole, bucloxic acid, carprofen, clidanac,diclofenac, diflunisal, diftalone, enolicam, etodolac, etoricoxib,fenbufen, fenclofenac, fenclozic acid, fenoprofen, fentiazac, feprazone,flufenamic acid, flufenisal, fluprofen, flurbiprofen, furofenac,ibufenac, ibuprofen, indomethacin, indoprofen, isoxepac, isoxicam,ketoprofen, ketorolac, lofemizole, lomoxicam, meclofenamate,meclofenamic acid, mefenamic acid, meloxicam, mesalamine, miroprofen,mofebutazone, nabumetone, naproxen, niflumic acid, oxaprozin, oxpinac,oxyphenbutazone, phenylbutazone, piroxicam, pirprofen, pranoprofen,salsalate, sudoxicam, sulfasalazine, sulindac, suprofen, tenoxicam,tiopinac, tiaprofenic acid, tioxaprofen, tolfenamic acid, tolmetin,triflumidate, zidometacin, zomepirac, and combinations thereof. In aparticular embodiment, the NSAID is selected from etodolac,flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meloxicam,naproxen, oxaprozin, piroxicam, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with an N-methyl d-aspartate (NMDA) receptor antagonist,examples of which include, by way of illustration and not limitation,including amantadine, dextromethorphan, dextropropoxyphene, ketamine,ketobemidone, memantine, methadone, and so forth.

In still another embodiment, compounds of the invention are administeredin combination with an opioid receptor agonist (also referred to asopioid analgesics). Representative opioid receptor agonists include, butare not limited to: buprenorphine, butorphanol, codeine, dihydrocodeine,fentanyl, hydrocodone, hydromorphone, levallorphan, levorphanol,meperidine, methadone, morphine, nalbuphine, nalmefene, nalorphine,naloxone, naltrexone, nalorphine, oxycodone, oxymorphone, pentazocine,propoxyphene, tramadol, and combinations thereof. In certainembodiments, the opioid receptor agonist is selected from codeine,dihydrocodeine, hydrocodone, hydromorphone, morphine, oxycodone,oxymorphone, tramadol, and combinations thereof.

In a particular embodiment, compounds of the invention are administeredin combination with a phosphodiesterase (PDE) inhibitor, particularly aPDE-V inhibitor. Representative PDE-V inhibitors include, but are notlimited to, avanafil, lodenafil, mirodenafil, sildenafil (Revatio®),tadalafil (Adcirca®), vardenafil (Levitra®), and udenafil.

In another embodiment, compounds of the invention are administered incombination with a prostaglandin analog (also referred to as prostanoidsor prostacyclin analogs). Representative prostaglandin analogs include,but are not limited to, beraprost sodium, bimatoprost, epoprostenol,iloprost, latanoprost, tafluprost, travoprost, and treprostinil, withbimatoprost, latanoprost, and tafluprost being of particular interest.

In yet another embodiment, compounds of the invention are administeredin combination with a prostaglandin receptor agonist, examples of whichinclude, but are not limited to, bimatoprost, latanoprost, travoprost,and so forth.

Compounds of the invention may also be administered in combination witha renin inhibitor, examples of which include, but are not limited to,aliskiren, enalkiren, remikiren, and combinations thereof.

In another embodiment, compounds of the invention are administered incombination with a selective serotonin reuptake inhibitor (SSRI).Representative SSRIs include, but are not limited to: citalopram and thecitalopram metabolite desmethylcitalopram, dapoxetine, escitalopram(e.g., escitalopram oxalate), fluoxetine and the fluoxetine desmethylmetabolite norfluoxetine, fluvoxamine (e.g., fluvoxamine maleate),paroxetine, sertraline and the sertraline metabolite demethylsertraline,and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a 5-HT_(1D) serotonin receptor agonist, examples ofwhich include, by way of illustration and not limitation, triptans suchas almotriptan, avitriptan, eletriptan, frovatriptan, naratriptanrizatriptan, sumatriptan, and zolmitriptan.

In one embodiment, compounds of the invention are administered incombination with a sodium channel blocker, examples of which include, byway of illustration and not limitation, carbamazepine, fosphenytoin,lamotrigine, lidocaine, mexiletine, oxcarbazepine, phenytoin, andcombinations thereof.

In one embodiment, compounds of the invention are administered incombination with a soluble guanylate cyclase stimulator or activator,examples of which include, but are not limited to ataciguat, riociguat,and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a tricyclic antidepressant (TCA), examples of whichinclude, by way of illustration and not limitation, amitriptyline,amitriptylinoxide, butriptyline, clomipramine, demexiptiline,desipramine, dibenzepin, dimetacrine, dosulepin, doxepin, imipramine,imipraminoxide, lofepramine, melitracen, metapramine, nitroxazepine,nortriptyline, noxiptiline, pipofezine, propizepine, protriptyline,quinupramine, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a vasopressin receptor antagonist, examples of whichinclude, by way of illustration and not limitation, conivaptan andtolvaptan.

Combined secondary therapeutic agents may also be helpful in furthercombination therapy with compounds of the invention. For example,compounds of the invention can be combined with a diuretic and an ARB,or a calcium channel blocker and an ARB, or a diuretic and an ACEinhibitor, or a calcium channel blocker and a statin. Specific examplesinclude, a combination of the ACE inhibitor enalapril (in the maleatesalt form) and the diuretic hydrochlorothiazide, which is sold under themark Vaseretic®, or a combination of the calcium channel blockeramlodipine (in the besylate salt form) and the ARB olmesartan (in themedoxomil prodrug form), or a combination of a calcium channel blockerand a statin, all may also be used with the compounds of the invention.Other therapeutic agents such as α₂-adrenergic receptor agonists andvasopressin receptor antagonists may also be helpful in combinationtherapy. Exemplary α₂-adrenergic receptor agonists include clonidine,dexmedetomidine, and guanfacine.

The following formulations illustrate representative pharmaceuticalcompositions of the invention.

Exemplary Hard Gelatin Capsules for Oral Administration

A compound of the invention (50 g), 440 g spray-dried lactose and 10 gmagnesium stearate are thoroughly blended. The resulting composition isthen loaded into hard gelatin capsules (500 mg of composition percapsule). Alternately, a compound of the invention (20 mg) is thoroughlyblended with starch (89 mg), microcrystalline cellulose (89 mg) andmagnesium stearate (2 mg). The mixture is then passed through a No. 45mesh U.S. sieve and loaded into a hard gelatin capsule (200 mg ofcomposition per capsule).

Alternately, a compound of the invention (30 g), a secondary agent (20g), 440 g spray-dried lactose and 10 g magnesium stearate are thoroughlyblended, and processed as described above.

Exemplary Gelatin Capsule Formulation for Oral Administration

A compound of the invention (100 mg) is thoroughly blended withpolyoxyethylene sorbitan monooleate (50 mg) and starch powder (250 mg).The mixture is then loaded into a gelatin capsule (400 mg of compositionper capsule). Alternately, a compound of the invention (70 mg) and asecondary agent (30 mg) are thoroughly blended with polyoxyethylenesorbitan monooleate (50 mg) and starch powder (250 mg), and theresulting mixture loaded into a gelatin capsule (400 mg of compositionper capsule).

Alternately, a compound of the invention (40 mg) is thoroughly blendedwith microcrystalline cellulose (Avicel PH 103; 259.2 mg) and magnesiumstearate (0.8 mg). The mixture is then loaded into a gelatin capsule(Size #1, White, Opaque) (300 mg of composition per capsule).

Exemplary Tablet Formulation for Oral Administration

A compound of the invention (10 mg), starch (45 mg) and microcrystallinecellulose (35 mg) are passed through a No. 20 mesh U.S. sieve and mixedthoroughly. The granules so produced are dried at 50-60° C. and passedthrough a No. 16 mesh U.S. sieve. A solution of polyvinylpyrrolidone (4mg as a 10% solution in sterile water) is mixed with sodiumcarboxymethyl starch (4.5 mg), magnesium stearate (0.5 mg), and talc (1mg), and this mixture is then passed through a No. 16 mesh U.S. sieve.The sodium carboxymethyl starch, magnesium stearate and talc are thenadded to the granules. After mixing, the mixture is compressed on atablet machine to afford a tablet weighing 100 mg.

Alternately, a compound of the invention (250 mg) is thoroughly blendedwith microcrystalline cellulose (400 mg), silicon dioxide fumed (10 mg),and stearic acid (5 mg). The mixture is then compressed to form tablets(665 mg of composition per tablet).

Alternately, a compound of the invention (400 mg) is thoroughly blendedwith cornstarch (50 mg), croscarmellose sodium (25 mg), lactose (120mg), and magnesium stearate (5 mg). The mixture is then compressed toform a single-scored tablet (600 mg of composition per tablet).

Alternately, a compound of the invention (100 mg) is thoroughly blendedwith cornstarch (100 mg) with an aqueous solution of gelatin (20 mg).The mixture is dried and ground to a fine powder. Microcrystallinecellulose (50 mg) and magnesium stearate (5 mg) are then admixed withthe gelatin formulation, granulated and the resulting mixture compressedto form tablets (100 mg of the compound of the invention per tablet).

Exemplary Suspension Formulation for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of the compound of the invention per 10 mL of suspension:

Ingredients Amount Compound of the invention 1.0 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum ® K(magnesium aluminum silicate) 1.0 g Flavoring 0.035 mL Colorings 0.5 mgDistilled water q.s. to 100 mL

Exemplary Liquid Formulation for Oral Administration

A suitable liquid formulation is one with a carboxylic acid-based buffersuch as citrate, lactate and maleate buffer solutions. For example, acompound of the invention (which may be pre-mixed with DMSO) is blendedwith a 100 mM ammonium citrate buffer and the pH adjusted to pH 5, or isblended with a 100 mM citric acid solution and the pH adjusted to pH 2.Such solutions may also include a solubilizing excipient such as acyclodextrin, for example the solution may include 10 wt %hydroxypropyl-β-cyclodextrin.

Other suitable formulations include a 5% NaHCO₃ solution, with orwithout cyclodextrin.

Exemplary Injectable Formulation for Administration by Injection

A compound of the invention (0.2 g) is blended with 0.4 M sodium acetatebuffer solution (2.0 mL). The pH of the resulting solution is adjustedto pH 4 using 0.5 N aqueous hydrochloric acid or 0.5 N aqueous sodiumhydroxide, as necessary, and then sufficient water for injection isadded to provide a total volume of 20 mL. The mixture is then filteredthrough a sterile filter (0.22 micron) to provide a sterile solutionsuitable for administration by injection.

Exemplary Compositions for Administration by Inhalation

A compound of the invention (0.2 mg) is micronized and then blended withlactose (25 mg). This blended mixture is then loaded into a gelatininhalation cartridge. The contents of the cartridge are administeredusing a dry powder inhaler, for example.

Alternately, a micronized compound of the invention (10 g) is dispersedin a solution prepared by dissolving lecithin (0.2 g) in demineralizedwater (200 mL). The resulting suspension is spray dried and thenmicronized to form a micronized composition comprising particles havinga mean diameter less than about 1.5 μm. The micronized composition isthen loaded into metered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 10 μgto about 500 μg of the compound of the invention per dose whenadministered by the inhaler.

Alternately, a compound of the invention (25 mg) is dissolved in citratebuffered (pH 5) isotonic saline (125 mL). The mixture is stirred andsonicated until the compound is dissolved. The pH of the solution ischecked and adjusted, if necessary, to pH 5 by slowly adding aqueous 1 NNaOH. The solution is administered using a nebulizer device thatprovides about 10 μg to about 500 μg of the compound of the inventionper dose.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of the invention. These specific embodiments,however, are not intended to limit the scope of the invention in any wayunless specifically indicated.

The following abbreviations have the following meanings unless otherwiseindicated and any other abbreviations used herein and not defined havetheir standard, generally accepted meaning:

-   -   AcOH acetic acid    -   BOC t-butoxycarbonyl (—C(O)OC(CH₃)₃)    -   (BOC)₂O di-t-butyl dicarbonate    -   CPME cyclopentyl methyl ether    -   DCC 1,3-dicyclohexylcarbodiimide    -   DCM dichloromethane or methylene chloride    -   DIPEA N,N-diisopropylethylamine    -   DMAP 4-dimethylaminopyridine    -   DMF N,N-dimethylformamide    -   EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide    -   Et₃N triethylamine    -   EtOAc ethyl acetate    -   HATU N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium        hexafluorophosphate    -   HCTU        (2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium        hexafluorophosphate)    -   HOBt 1-hydroxybenzotriazole    -   MeCN acetonitrile    -   MeOH methanol    -   MTBE methyl t-butyl ether    -   Pd(dppf)₂Cl₂ 1,1-bis(diphenylphosphino)ferrocene palladium        chloride    -   PE petroleum ether    -   THF tetrahydrofuran

Unless noted otherwise, all materials, such as reagents, startingmaterials and solvents, were purchased from commercial suppliers (suchas Sigma-Aldrich, Fluka Riedel-de Haen, and the like) and were usedwithout further purification.

Reactions were run under nitrogen atmosphere, unless noted otherwise.The progress of reactions were monitored by thin layer chromatography(TLC), analytical high performance liquid chromatography (anal. HPLC),and mass spectrometry, the details of which are given in specificexamples. Solvents used in analytical HPLC were as follows: solvent Awas 98% H₂O/2% MeCN/1.0 mL/L TFA; solvent B was 90% MeCN/10% H₂O/1.0mL/L TFA.

Reactions were worked up as described specifically in each preparationfor example; commonly reaction mixtures were purified by extraction andother purification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by preparative HPLC, typically using Microsorb C18and Microsorb BDS column packings and conventional eluents. Progress ofreactions was typically measured by liquid chromatography massspectrometry (LCMS). Characterization of isomers were done by NuclearOverhauser effect spectroscopy (NOE). Characterization of reactionproducts was routinely carried out by mass and ¹H-NMR spectrometry. ForNMR measurement, samples were dissolved in deuterated solvent (CD₃OD,CDCl₃, or DMSO-d₆), and ¹H-NMR spectra were acquired with a VarianGemini 2000 instrument (400 MHz) under standard observation conditions.Mass spectrometric identification of compounds was typically conductedusing an electrospray ionization method (ESMS) with an AppliedBiosystems (Foster City, Calif.) model API 150 EX instrument or anAgilent (Palo Alto, Calif.) model 1200 LC/MSD instrument.

Preparation 1: 1-Trityl-1H-1,2,3-triazole-4-carboxylic Acid

1H-1,2,3-Triazole-4-carboxylic acid (20.0 g, 177 mmol) was combined withDMF (200 mL, 2.6 mol) and pyridine (100 mL, 1.2 mol), and the resultingmixture was cooled to 0° C. Triphenylmethyl chloride (54 g, 190 mmol)was added in portions and the mixture was stirred at room temperaturefor 24 hours. The resulting slurry was filtered and the filter cake waswashed with water (2×200 mL) and air-dried yield an off white solid (60g). The solid was slurried in THF (800 mL) at room temperature for 4hours, then filtered. The filtrate was then concentrated by rotaryevaporation, yielding a thick oil. EtOAc (500 mL) was added and thevolume was reduced to ˜200 mL. The resulting thick slurry was filteredand dried to yield the title compound (35.5 g).

Preparation 2:(2S,4R)-5-Biphenyl-4-yl-4-t-butoxycarbonylamino-2-methyl-2-(tetrahydropyran-2-yloxymethyl)pentanoicAcid

(R)-3-Biphenyl-4-yl-2-t-butoxycarbonylamino-propionic acid (5.0 g, 15mmol) and 2,2-dimethyl-1,3-dioxane-4,6-dione (2.3 g, 16.1 mmol) werecombined in DMAP (3.2 g, 26.4 mmol). Additional DMAP (2.0 g, 16.1 mmol)and DCM (50 mL) was added and the resulting mixture was stirred andcooled to −5° C. (nitrogen purge) for 30 minutes. EDCI (HCl; (3.1 g,16.1 mmol) was added in portions, while maintaining the internaltemperature below 0° C. with stirring. The mixture was then cooled to−5° C., stirred at that temperature for 3 hours, then left at −20° C.overnight. The mixture was then washed with 0.4 M aqueous KHSO₄ (80 mL)and saturated aqueous NaCl (20 mL), then dried over MgSO₄ overnight. Thesolids were filtered off and the filtrate was then evaporated to drynessto yield crude Compound 1 (3.2 g).

AcOH (8.6 mL) was added to a solution of crude Compound 1 (6.4 g, 14mmol, 1.0 eq.) in anhydrous MeCN (90 mL) at −5° C. under nitrogen. Themixture was stirred at −5° C. for 30 minutes, then sodium borohydride(1.3 g, 34.5 mmol, 2.5 eq.) was added in small portions over 2 hours.After stirring for another 1 hour at −5° C., saturated aqueous NaCl and1.7 M of NaCl in water (30 mL) was added. The layers were separated andthe organic layer was washed with saturated aqueous NaCl (2×30 mL) andwater (2×30 mL), dried under MgSO₄, filtered and evaporated, Theresulting crude product was further purified by chromatography (5:1heptane:EtOAc) to yield Compound 2 (1.1 g, purity 98.4%) as a lightyellow solid.

Compound 2 (5.0 g, 11 mmol, 1.0 eq.) and K₂CO₃ (1.8 g, 13.2 mmol, 1.2eq.) were dissolved in DMF (33.9 mL) and cooled to 0° C. with stirringunder nitrogen. Methyl iodide (892 μL, 1.3 eq.) was added and theresulting mixture was stirred at 0° C. for 1 hour. The mixture wasallowed to warm to room temperature (23° C.) and held overnight.Saturated aqueous NaCl (35 mL) and EtOAc (35 mL) were added, and theresulting mixture was stirred for 2 minutes. The layers were separatedand the organic layer was evaporated. The residue was triturated withEtOAc (20 mL). The solid was filtered off and dried under vacuum. Thefiltrate was concentrated and triturated again with EtOAc to yield theCompound 3 (3.9 g),[(R)-2-biphenyl-4-yl-1-(2,2,5-trimethyl-4,6-dioxo-[1,3]dioxan-5-ylmethyl)ethyl]carbamicacid t-butyl ester.

Compound 3 (400.0 g, 855.5 mmol) was combined with CPME (2 L) to form aslurry. The slurry was cooled at 0° C. and 3.0 M HCl in CPME (2.0 L) wasadded. The resulting mixture was stirred at room temperature for 24hours, yielding a free flowing slurry. Filtration and drying yieldedCompound 4 as a 93:7 mixture of diastereoisomers (206 g total).Reslurrying in MeTHF (1 L) at room temperature followed by the additionof CPME (1 L; slurry overnight at room temperature) yielded Compound 5(170 g, de and purity 98%).

Compound 5 (25.0 g, 80.8 mmol) was combined with THF (500 mL) and NMM(25 mL, 230 mmol). The resulting mixture was cooled at 0° C. (jackettemp set at −5° C.) and isobutyl chloroformate (21.0 mL, 162 mmol) wasadded dropwise via addition funnel, while maintaining the internaltemperature below 5° C.). The mixture was stirred at 0° C. for 20minutes. Sodium borohydride (12.2 g, 323 mmol) dissolved in water (40mL) was added dropwise and the mixture was stirred at 0° C. for 20minutes (>98% conversion). The reaction was quenched with 1M aqueous HCl(300 mL) and the mixture was stirred at room temperature for 1 hour.Most of solvent was distilled off, leaving a white slurry. The slurrywas stirred for 60 minutes and then filtered (small particles, slowfiltration) to yield Compound 6 as a white solid (23 g, purity >98%).

Compound 6 (300 g, 1.0 mol) and DCM (3.8 L) were combined and theresulting mixture was cooled at 0° C. Dihydropyran (185 mL, 2.0 mol) andp-toluenesulfonic acid (52.5 g, 305 mmol) were added and the mixture wasstirred at room temperature for 2 hours. Aqueous saturated NaHCO₃(10:90, NaHCO₃:water, 3 L) was added and the phases were separated. Theorganic layer was dried with Na₂SO₄ followed by solvent removal toapproximately 500 mL. Into the crude product was added diisopropyl ether(2 L) and seed crystals. The resulting slurry was stirred overnight atroom temperature. Filtration and drying yielded crystalline Compound 7(320 g, purity >98%).

Compound 7 (320.0 g, 843.2 mmol) was dissolved in THF (2.5 L) to yield aclear solution, which was purged with nitrogen. The solution was cooledat 0° C. and 1.0 M NaHMDS in THF (920 mL, 920 mmol) was added dropwiseover 30 minutes. The mixture was stirred at 0° C. for 15 minutes thendi-t-butyldicarbonate (202 g, 926 mmol) dissolved in THF (500 mL) wasadded dropwise over 1 hour, while maintaining the internal temperaturebelow 5° C. The mixture was allowed to warm to room temperature (>99%conversion to Compound 8). The mixture was cooled to <5° C. followed bythe addition of 1.0 M aqueous LiOH (2.5 L, 2.5 mol). The cooling bathwas removed and the mixture was stirred overnight at 27° C. (˜4%starting material remaining). The mixture was heated at 35° C. for 4hours (>98% conversion), then cooled to 15° C. The mixture was dilutedwith EtOAc (3 L) and saturated aqueous NH₄Cl (0.37:0.63, NH₄Cl:water, 3L). The phases were separated, and the organic layer was washed withsaturated aqueous NH₄Cl (3 L) and saturated aqueous NaCl (3 L). Theorganic layer dried with Na₂SO₄ (1 kg), followed by solvent removal toyield the crude title compound (463 g) as a glassy sticky solid.

Preparation 3:(2S,4R)-5-Biphenyl-4-yl-2-methyl-2-(tetrahydropyran-2-yloxymethyl)-4-[(1-trityl-1H-1,2,3-triazole-4-carbonyl)amino]pentanoicAcid

(2S,4R)-5-Biphenyl-4-yl-4-t-butoxycarbonylamino-2-methyl-2-(tetrahydropyran-2-yloxymethyl)pentanoicacid (10.0 g, 20.1 mmol) was combined with DMF (50 mL, 600 mmol), andstirred. K₂CO₃ (3.3 g, 24 mmol) was added and the resulting mixture wascooled to 0° C. Benzyl bromide (3.0 mL, 25 mmol) was added and themixture was stirred from 0° C. to room temperature, and then overnight.1.0 M HCl in water (250 mL, 250 mmol) and EtOAc (300 mL, 3.0 mol) wereadded. The phases were separated and the organic layer was washed withsaturated aqueous NaCl (200 mL) and dried over Na₂SO₄, followed bysolvent removal. DCM (50 mL) and 3.0 M HCl in CPME (100 mL, 300 mmol)were added and the resulting mixture was stirred at room temperatureovernight. The volume was reduced by half by rotary evaporation,yielding a free-flowing slurry, which was filtered. The flask and filtercake were washed with CPME (20 mL) and dried. The residue was dissolvedin DCM (50 mL, 800 mmol) and resulting suspension was cooled at 0° C. to10° C. Dihydropyran (3.7 mL, 40.2 mmol) and p-toluenesulfonic acid (692mg, 4.0 mmol) were added and the resulting mixture was stirred at 0° C.for 2 hours, then stirred overnight at a cool temperature. The volumewas reduced to −20 mL by rotary evaporation. MTBE was added (˜30 mL)followed by seed crystals, yielding a thin slurry after 15 minutes ofstirring. The volume was reduced by half and additional MTBE (20 mL) wasadded, while stirring at room temperature to yield a thick slurry.Additional MTBE (to 100 mL volume) was added and the mixture was stirredfor 1 hour. Filtration and drying yielded Compound 1 (8.9 g) as an HClsalt.

1-Trityl-1H-1,2,3-triazole-4-carboxylic acid (9.2 g, 26 mmol) wasdissolved in THF (200 mL, 2.0 mol). DIPEA (9.0 mL, 52 mmol) was addedand the resulting mixture was cooled to 0° C. HCTU (11 g, 26 mmol) wasadded in portions and mixture was stirred at 0° C. for 15 minutes.Compound 1 (HCl salt; 9.0 g, 17 mmol) was added and the resultingmixture was stirred from 0° C. to room temperature. The reaction wasmonitored and quenched with water (200 mL) after 90 minutes. EtOAc (200mL) was added. The organic layer was washed with saturated aqueous NaCl(200 mL), dried over Na₂SO₄, and the solvent removed. The residue (15 g)was dissolved in DCM (100 mL), the solids were filtered off and theclear solution was purified (300 g SiG column; elution with 10-30% EtOAcin hexanes) to yield Compound 2 (7.5 g).

Compound 2 (0.20 g, 0.24 mmol) was combined with EtOAc (3 mL, 30 mmol).NaHCO₃ (50 mg, 0.6 mmol) was added and the resulting clear solution waspurged with nitrogen. 10% Pd/C (0.05:0.45, Palladium:carbon black, 50mg, 0.05 mmol) was added and the resulting mixture was purged withhydrogen and then hydrogenated overnight at room temperature. The solidswere filtered off and the solvent was removed by rotary evaporation toyield the title compound.

Preparation 4:(2S,4R)-5-Biphenyl-4-yl-2-methyl-2-(tetrahydropyran-2-yloxymethyl)-4-[(1H-1,2,3-triazole-4-carbonyl)amino]pentanoicAcid

(2S,4R)-5-Biphenyl-4-yl-2-methyl-2-(tetrahydropyran-2-yloxymethyl)-4-[(1-trityl-1H-1,2,3-triazole-4-carbonyl)amino]pentanoicacid benzyl ester (7.5 g, 9.1 mmol) was combined with EtOAc (80 mL, 800mmol). The resulting clear solution was purged with nitrogen and 10%Pd/C (0.05:0.45, Palladium:carbon black, 1.0 g, 0.94 mmol) was added.The resulting mixture was purged with hydrogen and then hydrogenatedovernight at room temperature. The mixture was purged with nitrogen, thesolids were filtered off, and the solvent was removed by rotaryevaporation to yield the title compound (7 g).

Preparation 5: (R)-3-(4-Bromophenyl)-2-t-butoxycarbonylaminopropionicAcid

To a solution of (R)-2-amino-3-(4-bromophenyl)propionic acid (50 g, 0.2mol) in MeCN (700 mL) was added a solution of NaOH (16.4 g, 0.4 mol) inwater (700 mL) at −5° C. After stirring for 10 minutes, a solution of(BOC)₂O (44.7 g, 0.2 mol) in MeCN (100 mL) was added. The mixture waswarmed to room temperature and stirred overnight. After evaporation ofthe MeCN, the residue was diluted with DCM (800 mL) and acidified with 1M HCl to pH 2 at −5° C. The aqueous layer was extracted with DCM (3×200mL). The combined organic layers were washed with saturated aqueous NaCl(500 mL), dried over anhydrous Na₂SO₄ and concentrated to yield thetitle compound as a white solid (64.2 g). LC-MS: [M+Na]: 366, [2M+Na]:709.

Preparation 6:[(R)-1-(3′-Fluorobiphenyl-4-ylmethyl)-2-(2,2,5-trimethyl-4,6-dioxo-[1,3]dioxan-5-yl)ethyl]carbamicAcid t-Butyl Ester

To a solution of (R)-3-(4-bromophenyl)-2-t-butoxycarbonylaminopropionicacid (64.2 g, 187 mmol) in 1,4-dioxane (500 mL) was added3-fluorophenylboronic acid (31.3 g, 224 mmol) and Pd(dppf)₂Cl₂ (13.7 g,19 mmol) at room temperature under nitrogen. After stirring for 10minutes, a solution of K₂CO₃ (51.7 g, 374 mmol) in water (250 mL) wasadded. The mixture was heated to 100° C. and stirred overnight. Afterevaporation of the solvent, water (200 mL) was added. The aqueous layerwas acidified with 1 M HCl to pH 2 and extracted with EtOAc (3×200 mL).The combined organic layers were washed with saturated aqueous NaCl (400mL), dried over anhydrous Na₂SO₄, and concentrated to yield the crudeproduct which was further purified by column chromatography(hexanes:EtOAc=4:1) to yield Compound 1 as a light yellow oil (45 g).LC-MS: [M+Na]: 382, [2M+Na]: 741.

To a solution of Compound 1 (45 g, 125 mmol), Meldrum's acid (23.5 g,163 mmol), and DMAP (26.0 g, 213 mmol) in anhydrous DCM (500 mL) wasadded a solution of DCC (33.3 g, 163 mmol) in anhydrous DCM (200 mL)over 1 hour at −5° C. under nitrogen. The mixture was stirred at −5° C.for 8 hours, then refrigerated overnight, during which tiny crystals ofdicyclohexylurea precipitated. After filtration, the mixture was washedwith 5% KHSO₄ (4×200 mL) and saturated aqueous NaCl (1×200 mL), thendried under refrigeration with anhydrous MgSO₄ overnight. The solutionwas evaporated to yield the crude Compound 2 as a light yellow oil (57.7g). LC-MS: [M+Na]: 508, [2M+Na]: 993.

To a solution of Compound 2 (57.7 g, 119 mmol) in anhydrous DCM (1 L)was added AcOH (78.4 g, 1.3 mol) at −5° C. under nitrogen. The mixturewas stirred at −5° C. for 0.5 hour, then NaBH₄ (11.3 g, 0.3 mol) wasadded in small portions over 1 hour. After stirring for an additional 1hour at −5° C., saturated aqueous NaCl (300 mL) was added. The organiclayer was washed with saturated aqueous NaCl (2×300 mL) and water (2×300mL), dried over anhydrous MgSO₄, filtered and concentrated to yield thecrude product, which was further purified by chromatography(hexanes:EtOAc=6:1) to yield Compound 3 as a light yellow oil (28 g).LC-MS: [M+Na]: 494, [2M+Na]: 965.

To a solution of Compound 3 (28 g, 60 mmol) in anhydrous DMF (250 mL)was added K₂CO₃ (9.9 g, 72 mmol) and methyl iodide (25.6 g, 180 mmol) at0° C. under nitrogen. After stirring for 1 hour at 0° C., the mixturewas warmed to room temperature and stirred overnight. The mixture wasdiluted with water (3 L) and extracted with EtOAc (3×300 mL). Thecombined organic layers were washed with saturated aqueous NaCl (500mL), dried over anhydrous Na₂SO₄, and concentrated to give the crudeproduct which was further purified by chromatography (hexanes:EtOAc=5:1)to yield the title compound as a light yellow solid (11.7 g). LC-MS:[M+Na]=508, [2M+Na]=993. ¹H NMR (300 MHz, CD₃OD): δ7.52-7.49 (m, 2H),7.41-7.39 (m, 2H), 7.32-7.27 (m, 3H), 7.07-7.01 (m, 1H), 6.21-6.18 (d,1H), 3.79 (m, 1H), 2.78-2.61 (m, 2H), 2.35-2.20 (m, 2H), 1.76 (s, 6H),1.59 (s, 3H), 2.21 (s, 1H), 1.28 (s, 9H).

Preparation 7:(2S,4R)-4-t-Butoxycarbonylamino-5-(3′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-pentanoicAcid (Compound 1) and(2S,4R)-4-Amino-5-(3′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-pentanoicAcid (Compound 2)

Distilled Water (181 mL) was purged 1 hour under nitrogen, thencannulated into a vessel containing 0.1 M of samarium diiodide in THF(800 mL). While maintaining an atmosphere of nitrogen, a similarlydegassed solution of[(R)-1-(3′-fluorobiphenyl-4-ylmethyl)-2-(2,2,5-trimethyl-4,6-dioxo-[1,3]dioxan-5-yl)ethyl]carbamicacid t-butyl ester (4.9 g, 10.0 mmol, 1.0 eq.) and THF (20 mL) was addedvia canula. The resulting mixture was stirred for 15 minutes, thenexposed to air. The solvent was evaporated, and EtOAc (200 mL),saturated aqueous NaCl (50 mL) and 10% citric acid (20 mL) were added.The mixture was stirred for 5 minutes, then both layers were extracted.The organic layer was dried over Na₂SO₄ and concentrated under vacuum.The crude product was purified by chromatography (330 g gold column, 1:1ether:EtOAc with 0.5% AcOH) to yield title Compound 1 (1.5 g). A portionof Compound 1 was dissolved in 4M HCl in dioxane (6 mL) and MeCN (10mL). The solvent was evaporated under vacuum to yield title Compound 2.

Preparation 8:[(R)-1-(4-Bromobenzyl)-2-(2,2,5-trimethyl-4,6-dioxo-[1,3]dioxan-5-yl)ethyl]carbamicAcid t-Butyl Ester

To a mixture of (R)-2-amino-3-(4-bromophenyl)propionic acid (100 g, 410μmol) in MeCN (600 mL) was added dropwise a solution of NaOH (32.8 g,820 μmol) in water (800 mL) at 0° C. The resulting solution was stirredfor 30 minutes. A solution of (BOC)₂O (93.8 g, 430 μmol) in MeCN (200mL) was added, and the resulting mixture was warmed to room temperatureand stirred overnight. The MeCN was evaporated and the residue wasdiluted with DCM (1 L) and acidified with 2 M HCl to pH=2 at −5° C. Theaqueous was extracted and the combined organic layers were washed withsaturated aqueous NaCl (500 mL), dried over anhydrous Na₂SO₄ andconcentrated to yield crude Compound 1 (141 g, 100%) as a yellow solid.LC-MS: 366[M+Na]⁺.

Compound 1 (20 g, 58.1 mmol) was combined with2,2-dimethyl-1,3-dioxane-4,6-dione (9.2 g, 63.9 mmol), DMAP (10.7 g,87.2 mmol), and anhydrous DCM (400 mL), and cooled to 0° C. Afterstirring for 30 minutes, a solution of DCC (13.2 g, 63.9 mmol) in DCM(50 mL) was added dropwise at 0° C. under nitrogen. After the addition,the ice bath was removed and the mixture was stirred at room temperatureovernight. The solution was cooled at −20° C. for 1 hour and then thesolids were filtered off. The filtrate was washed with a 5% KHSO₄solution (4×100 mL) and saturated aqueous NaCl (200 mL). The organiclayer was dried over anhydrous Na₂SO₄ and evaporated to yield crudeCompound 2 (27.5 g) as a gray solid. LC-MS: 492 [M+Na]⁺.

To a solution of Compound 2 (27.5 g, 58.1 mmol) in anhydrous DCM (400mL) was added AcOH (38.4 g, 639.1 mmol) at −5° C. under nitrogen. Themixture was stirred at −5° C. for 30 minutes. NaBH₄ (5.5 g, 145.2 mmol)was added in portions over 30 minutes, and the resulting solution wasstirred at room temperature for 3 hours. Saturated aqueous NaCl (300 mL)was added to quench the reaction. The organic layer was washed withsaturated aqueous NaCl (2×200 mL), dried over anhydrous Na₂SO₄ andconcentrated to yield crude Compound 3 (22.6 g). LC-MS: 478 [M+Na]⁺.

To a solution of Compound 3 (22.6 g, 49.6 mmol) and K₂CO₃ (8.3 g, 59.5mmol) in anhydrous DMF (160 mL) was added methyl iodide (14 g, 99.2mmol) dropwise at 0° C. After the addition, the solution was stirred atroom temperature overnight. The mixture was evaporated and the residuewas dissolved in EtOAc (500 mL) and washed with saturated aqueous NaCl(2×200 mL). The organic solution was dried over anhydrous Na₂SO₄ andconcentrated to yield the crude product which was triturated with ethylether (100 mL), then filtered to yield the title compound (14.5 g) as awhite solid. LC-MS: 492 [M+Na]⁺.

Preparation 9:(2S,4R)-4-t-Butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicAcid

A mixture of[(R)-1-(4-bromobenzyl)-2-(2,2,5-trimethyl-4,6-dioxo-[1,3]dioxan-5-yl)ethyl]carbamicacid t-butyl ester (8 g, 17 mmol), 3-chlorophenylboronic acid (3 g, 18.7mmol), Pd(dppf)₂Cl₂ (400 mg, 550 μmol) and potassium fluoride (2 g, 34mmol) in water (80 mL) and dioxane (80 mL) was stirred at 60° C. underargon for 3 hours. The mixture was concentrated, dispersed in water (150mL), extracted with EtOAc (2×100 mL), dried over anhydrous Na₂SO₄ andevaporated to yield the crude product, which was purified by columnchromatography (PE:EtOAc=10:1) to yield Compound 1 (7 g) as a whitesolid. LC-MS: 524 [M+Na]⁺

Samarium powder (50 g, 330 μmol) was flushed with argon (20 minutes).Anhydrous THF (1.5 L) was added and the resulting suspension was bubbledwith argon (15 minutes). Iodine (70 g, 270 mmol) was added and themixture was flushed again with argon (10 minutes). The mixture wascovered with aluminium foil and heated at 65° C. overnight then allowedto cool to room temperature. A solution of Compound 1 (7 g, 13.9 mmol)in THF (200 mL) and water (100 mL) was sealed and flushed with argon (10minutes), cooled to −70° C., flushed with argon (10 minutes), cooled to−70° C., and flushed with argon (30 minutes). The samarium powdersolution (1.5 L) was then added to the cooled solution via cannula, andstirred at room temperature for 2 hours. The solution was evaporated,and the residue was dissolved in EtOAc (200 mL), washed with tartaricacid solution (10%, 150 mL), dried over anhydrous Na₂SO₄, concentratedand purified by column chromatography (PE:EtOAc=0 to 30%, added with0.05% AcOH) to yield the title compound (3 g) as a white solid. LC-MS:470 [M+Na]⁺. ¹H NMR (300 MHz, CD3OD): δ 7.28˜7.56 (m, 8H), 3.94 (s, 1H),3.56˜3.66 (m, 2H), 2.69˜2.82 (m, 2H), 1.70˜1.90 (m, 2H), 1.17˜1.31 (m,12H).

Preparation 10:(2S,4R)-4-t-Butoxycarbonylamino-5-(2′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicAcid

A mixture of[(R)-1-(4-bromobenzyl)-2-(2,2,5-trimethyl-4,6-dioxo-[1,3]dioxan-5-yl)ethyl]carbamicacid t-butyl ester (4.8 g, 30.6 mmol), 2-chlorophenylboronic acid,Pd(dppf)₂Cl₂ (1.0 g, 1.3 mmol) and potassium fluoride (2.9 g, 51 mmol)in water (50 mL) and dioxane (250 mL) was stirred at 60° C. under argonfor 3 hours. The mixture was concentrated, dissolved in water (150 mL),extracted with EtOAc (2×200 mL), dried over anhydrous Na₂SO₄ andevaporated to yield the crude product, which was purified by columnchromatography (PE:EtOAc=3:1) to yield Compound 1 (10 g) as a whitesolid. LC-MS: 402[M-Boc]⁺. ¹H NMR (300 MHz, CDCl₃): δ 7.47 (m, 1H), 7.38(d, J=8.0 Hz, 2H), 7.31 (m, 3H), 7.23 (dd, J=9.9, 5.7 Hz, 2H), 4.18 (d,J=10.2 Hz, 1H), 4.01 (s, 1H), 2.87 (dd, J=13.8, 5.7 Hz, 1H), 2.71 (dd,J=13.7, 6.6 Hz, 1H), 2.30 (m, 2H), 1.75 (s, 6H), 1.65 (s, 3H), 1.33 (d,J=11.7 Hz, 9H).

Samarium powder (50 g, 330 μmol) was flushed with argon (20 minutes).Anhydrous THF (1.5 L) was added and the resulting suspension was bubbledwith argon (15 minutes). Iodine (70 g, 270 mmol) was added and themixture was flushed again with argon (10 minutes). The mixture wascovered with aluminium foil and heated at 65° C. overnight then allowedto cool to room temperature. A solution of Compound 1 (7 g, 13.9 mmol)in THF (200 mL) and water (100 mL) was sealed and flushed with argon (10minutes), cooled to −70° C., flushed with argon (10 minutes), cooled to−70° C., and flushed with argon (30 minutes). The samarium powdersolution (1.5 L) was then added to the cooled solution via cannula, andstirred at room temperature for 2 hours. The solution was evaporated,and the residue was dissolved in EtOAc (200 mL), washed with tartaricacid solution (10%, 150 mL), dried over anhydrous Na₂SO₄, concentratedand purified by column chromatography (PE:EtOAc=0 to 30%, added with0.05% AcOH) to yield the title compound (2.8 g) as an off-white solid.LC-MS: 348[M-Boc]⁺. ¹H NMR (300 MHz, CD₃OD): δ 7.46 (m, 1H), 7.28 (m,7H), 3.97 (s, 1H), 3.63 (m, 2H), 2.82 (m, 1H), 2.69 (m, 1H), 1.89 (m,1H), 1.74 (m, 1H), 1.33 (m, 7H), 1.22 (m, 5H).

Preparation 11:(2S,4R)-4-t-Butoxycarbonylamino-5-(2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicAcid

A mixture of[(R)-1-(4-bromobenzyl)-2-(2,2,5-trimethyl-4,6-dioxo-[1,3]dioxan-5-yl)ethyl]carbamicacid t-butyl ester (12 g, 25.6 mmol), 2-fluorophenylboronic acid (4.3 g,30.7 mmol), Pd(dppf)₂Cl₂ (950 mg, 1.3 mmol) and potassium fluoride (3.0g, 51.2 mmol) in water (50 mL) and dioxane (100 mL) was stirred at 60°C. under argon for 2 hours. The mixture was concentrated, diluted withwater (100 mL), extracted with EtOAc (3×100 mL), dried over anhydrousNa₂SO₄ and evaporated to yield the crude product, which was purified bycolumn chromatography (PE:EtOAc=3:1) to yield Compound 1 (10 g). LC-MS:386.1 [M-Boc]⁺. ¹H NMR (300 MHz, CDCl₃): δ 7.43 (m, 3H), 7.21 (m, 6H),4.15 (d, J=10.6 Hz, 1H), 3.99 (s, 1H), 2.83 (m, 1H), 2.70 (dd, J=13.8,6.8 Hz, 1H), 2.26 (m, 2H), 1.74 (s, 6H), 1.63 (s, 3H), 1.27 (m, 9H).

Samarium powder (50 g, 330 μmol) was flushed with argon (20 minutes).Anhydrous THF (1.5 L) was added and the resulting suspension was bubbledwith argon (15 minutes). Iodine (70 g, 270 mmol) was added and themixture was flushed again with argon (10 minutes). The mixture wascovered with aluminium foil and heated at 65° C. overnight then allowedto cool to room temperature. A solution of Compound 1 (7 g, 14.4 mmol)in THF (200 mL) and water (100 mL) was sealed and flushed with argon (10minutes), cooled to −70° C., flushed with argon (10 minutes), cooled to−70° C., and flushed with argon (30 minutes). The samarium powdersolution (1.5 L) was then added to the cooled solution via syringe, andstirred at room temperature for 2 hours. The solution was evaporated,and the residue was dissolved in EtOAc (200 mL), washed with tartaricacid solution (10%, 150 mL), dried over anhydrous Na₂SO₄, concentratedand purified by column chromatography (PE:EtOAc=0 to 30%, added with0.05% AcOH) to yield the title compound (2.6 g) as an off-white solid.LC-MS: 332.0[M-Boc]⁺. ¹H-NMR (CD₃OD, 300 Hz): δ 7.29 (m, 8H), 3.96 (s,1H), 3.62 (m, 2H), 2.81 (m, 1H), 2.68 (m, 1H), 1.89 (m, 1H), 1.73 (m,1H), 1.31 (m, 7H), 1.23 (m, 5H).

Preparation 12:(2S,4R)-4-Amino-5-(2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicAcid

(2S,4R)-4-t-Butoxycarbonylamino-5-(2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (114 mg, 265 μmol) was combined with DIPEA (3 eq.) in DMF (0.2 mL)to yield the title compound.

Preparation 13:(2S,4R)-5-(4-bromophenyl)-4-t-butoxycarbonylamino)-2-(hydroxymethyl)-2-methylpentanoicAcid

Samarium powder (32 g, 210 mmol) was added to an oven dried flask, andthe flask was sealed and flushed with argon for 20 minutes. AnhydrousTHF (800 mL) was added and the resulting suspension was bubbled withargon for 15 minutes. Iodine (44.8 g, 176 mmol) was added and the flaskflushed again with argon for 10 minutes. The flask was covered andheated at 65° C. overnight, then allowed to cool to room temperature.The resulting SmI₂ solution was used directly in the next step.

A solution of[(R)-1-(4-bromobenzyl)-2-(2,2,5-trimethyl-4,6-dioxo-[1,3]dioxan-5-yl)ethyl]carbamicacid t-butyl ester (4 g, 8.5 mmol) in THF (200 mL) and water (100 mL)was sealed and flushed with argon for 10 mins, then cooled to −70° C.and flushed with argon for another 10 minutes, then again cooled to −70°C. and flushed with argon for another 30 minutes. The SmI₂ solution (800mL) was then added and the resulting solution was stirred at roomtemperature for 2 hours. The solution was evaporated, diluted with EtOAc(200 mL), washed with a tartaric acid solution (10%, 150 mL), dried,concentrated and purified by column chromatography (PE:EA=0 to 30%,added with 0.05% acetic acid) to yield the title compound (1.7 g) as anoff-white solid. LC-MS: [M-Boc]⁺: 316. ¹H NMR (300 MHz, CD₃OD): δ 7.36(m, 2H), 7.12 (m, 2H), 3.97 (s, 1H), 3.60 (m, 2H), 2.6˜2.7 (m, 2H),1.69˜1.81 (m, 2H), 1.15˜1.37 (m, 12H).

Preparation 14:(2S,4R)-5-(4-Bromophenyl)-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

(2S,4R)-5-(4-bromophenyl)-4-t-butoxycarbonylamino)-2-(hydroxymethyl)-2-methylpentanoicacid (1.0 g, 2.4 mmol) was combined with MeCN (20 mL). 4N HCl in dioxane1.8 mL, 7.2 mmol) was added. The resulting mixture was stirred for 30minutes then concentrated under reduced pressure.

1H-[1,2,3]Triazole-4-carboxylic acid (272 mg, 2.4 mmol) and HATU (959mg, 2.5 mmol) were combined in DMF (2 mL) and stirred for 10 minutes.DIPEA (1.3 mL, 7.2 mmol) and Compound 1 in DMF (2 mL) were added and theresulting mixture was stirred for 30 minutes then concentrated. Theresidue was purified by reverse phase chromatography (20-100% MeCN inwater) to yield the title compound (287 mg).

Preparation 15:(2S,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicAcid Ethyl Ester (Compound 2) and(2S,4R)-4-t-Butoxycarbonylamino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicAcid Ethyl Ester (Compound 3)

(2S,4R)-5-(4-bromophenyl)-4-((t-butoxycarbonyl)amino)-2-(hydroxymethyl)-2-methylpentanoicacid (1.3 mg, 3.1 mmol) was combined with 5-chloro-2-fluorophenylboronicacid (708 mg, 4.1 mmol), sodium carbonate (993 mg, 9.4 mmol), water (0.2mL) and dioxane (1.5 mL). The reaction vessel was sealed, air wasremoved by vacuum, and the vessel was purged with nitrogen.Tetrakis(triphenylphosphine)palladium (0) (541 mg, 468 μmol) was quicklyadded and air was removed by vacuum. The mixture was heated at 90° C.for 45 minutes. The mixture was acidified with 1N HCl/water to pH ˜4,then extracted with EtOAc. The solvent was removed and the residue wasdissolved in AcOH and purified by reverse phase chromatography to yieldCompound 1.

Compound 1 (1.0 g, 2.1 mmol) was dissolved in EtOH (4 mL) and 4N HCl indioxane (4 mL) and stirred for 3 hours at 60° C. The solvent wasevaporated to yield crude Compound 2, which was carried directly to thenext step.

Compound 2 (800 mg, 2.0 mmol) was dissolved in DCM and (BOC)₂O (472 μl,2.0 mmol), followed by the addition of Et₃N (566 μL, 4.1 mmol) and DMAP(1 flake). The resulting mixture was stirred for 3 hours. The solventwas removed and the crude product was triturated with DCM and filteredto yield Compound 3 (800 g), which was used without furtherpurification.

Preparation 16:(2S,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicAcid

The title compound can be prepared by deprotection of(2S,4R)-4-amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid ethyl ester.

Preparation 17:(2S,4R)-5-Biphenyl-4-yl-4-t-butoxycarbonylamino-2-hydroxymethyl-2-methylpentanoicAcid (P²═BOC) and(2S,4R)-4-Amino-5-biphenyl-4-yl-2-hydroxymethyl-2-methylpentanoic Acid(P² Removed)

Distilled water (140 mL) was purged 30 minutes under nitrogen, thencannulated into a vessel containing 0.1 M of samarium diiodide in THF(800 mL), exercising caution not to allow any air to come into contactwith solution. While maintaining an atmosphere of nitrogen, a degassedsolution of[(R)-2-biphenyl-4-yl-1-(2,2,5-trimethyl-4,6-dioxo-1,3-dioxinan-5-ylmethyl)ethyl]carbamicacid t-butyl ester (3.7 g, 8.0 mmol, 1.0 eq.) and THF (100 mL) was addedvia canula. The resulting mixture was stirred for 15 minutes, thenexposed to air. Saturated aqueous NaCl (12 mL), 10% citric acid (6 mL),and EtOAc (30 mL) were added. The mixture was stirred for 5 minutes,then both layers were extracted. The organic layer was dried over Na₂SO₄and concentrated under vacuum. The crude product was purified bychromatography (330 g gold column, 50% EtOAc with 0.5% AcOH/ethergradient) to yield the BOC-protected acid (P²═BOC) (1.4 g). TheBOC-protected acid was dissolved in MeCN (10 mL), followed by theaddition of 4N HCl in dioxane (10 mL). The solvent was evaporated andthe product azeotroped with toluene (2×) to yield the acid. (P² removed)(1.0 g).

Preparation 18:(2S,4R)-4-Amino-5-biphenyl-4-yl-2-methoxymethyl-2-methylpentanoic AcidEthyl Ester

(2S,4R)-5-Biphenyl-4-yl-4-t-butoxycarbonylamino-2-hydroxymethyl-2-methylpentanoicacid ethyl ester (100 mg, 226 μmol) and tetrabutylammonium hydrogensulfate (15 mg, 45 μmol) were combined with DCM (1 mL) and NaOH (159 μL,1.6 mmol). Dimethyl sulfate (114 mg, 906 μmol) was added and thereaction vessel was sealed and stirred vigorously overnight. The mixturewas then concentrated under reduced pressure and the residue wasdissolved in AcOH and purified by reverse phase chromatography (30-100%MeCN in water) to yield Compound 1 (30 mg).

Compound 1 (30 mg, 66 μmol) was combined with MeCN (1 mL) and 4N HCl indioxane (0.3 mL) and stirred for 10 minutes, then concentrated underreduced pressure to yield the title compound (23 mg).

Preparation 19:(2S,4R)-4-t-Butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicAcid Ethyl Ester

(2S,4R)-4-t-Butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (860 mg, 1.9 mmol) was dissolved in EtOH (4 mL) and 4N HCl indioxane (4 mL) and stirred for 3 hours at 60° C. The solvent wasevaporated and the crude Compound 1 was carried to next step.

Compound 1 (722 mg, 1.9 mmol) was dissolved in DCM and (BOC)₂O (446 μL,1.9 mmol). Et₃N (535 μL, 3.8 mmol) and DMAP (1 flake) were added and theresulting mixture was stirred for 3 hours. The solvent was evaporatedand the residue was purified (normal phase chromatography 0-60%EtOAc/hexanes) to yield the title compound (800 mg).

Preparation 20:(2S,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-methoxymethyl-2-methylpentanoicAcid Ethyl Ester

(2S,4R)-4-t-Butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid ethyl ester (100 mg, 226 μmol) and tetrabutylammonium hydrogensulfate (15 mg, 45 μmol) were combined with DCM (1 mL) and NaOH (159 μL,1.6 mmol). Dimethyl sulfate (114 mg, 906 μmol) was added and thereaction vessel was sealed and stirred vigorously overnight. The mixturewas then concentrated under reduced pressure and the residue wasdissolved in AcOH and purified by reverse phase chromatography (30-100%MeCN in water) to yield Compound 1 (32 mg).

Compound 1 (32 mg, 66 μmol) was combined with MeCN (1 mL) and 4N HCl indioxane (0.3 mL) and stirred for 10 minutes, then concentrated underreduced pressure to yield the title compound (26 mg).

Preparation 21:(2S,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-methoxymethyl-2-methylpentanoicAcid Ethyl Ester

(2S,4R)-4-t-Butoxycarbonylamino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-pentanoicacid ethyl ester (415 mg, 840 μmol) and tetrabutylammonium hydrogensulfate (57 mg, 168 μmol) were combined with DCM (1 mL) and NaOH (588μL, 5.9 mmol). Dimethyl sulfate (424 mg, 3.4 mmol) was added and thereaction vessel was sealed and stirred vigorously overnight. The mixturewas extracted with DCM and water, purified (normal phase chromatography;0-60 EtOAc:hexanes), and concentrated under reduced pressure to yieldCompound 1 (220 mg).

Compound 1 (88 mg, 173 μmol) was combined with MeCN (1 mL) and 4N HCl indioxane (0.3 mL) and stirred for 10 minutes, then concentrated underreduced pressure to yield the title compound (34 mg).

Preparation 22:(2S,4R)-4-Amino-5-(5′-chloro-2′-fluoro-biphenyl-4-yl)-2-ethoxymethyl-2-methylpentanoicAcid Ethyl Ester

(2S,4R)-4-t-Butoxycarbonylamino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-pentanoicacid ethyl ester (415 mg, 840 μmol) and tetrabutylammonium hydrogensulfate (57 mg, 168 μmol) were combined with DCM (1 mL) and NaOH (588μL, 5.9 mmol). Diethyl sulfate (518 mg, 3.4 mmol) was added and thereaction vessel was sealed and stirred vigorously overnight. The mixturewas extracted with DCM and water, purified (normal phase chromatography;0-60 EtOAc:hexanes), and concentrated under reduced pressure to yieldCompound 1 (110 mg).

Compound 1 (90 mg, 173 μmol) was combined with MeCN (1 mL) and 4N HCl indioxane (0.3 mL) and stirred for 10 minutes, then concentrated underreduced pressure to yield the title compound (35.2 mg).

Example 1

It is understood that the compounds of Example 1 can exist in a tautomerform, and that both forms are covered by this example. For example,(2S,4R)-5-biphenyl-4-yl-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)-amino]pentanoicacid 5-t-Butyl-2-oxo-[1,3]dioxol-4-ylmethyl ester is depicted in Example1A but it is understood that this compound can exist in a tautomer form,for example, as(2S,4R)-5-biphenyl-4-yl-2-hydroxymethyl-2-methyl-4-[(3H-[1,2,3]triazole-4-carbonyl)-amino]-pentanoicacid 5-t-butyl-2-oxo-[1,3]dioxol-4-ylmethyl ester. The same is true forthe compounds in Examples 1B-1J.

1A:(2S,4R)-5-Biphenyl-4-yl-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)-amino]pentanoicAcid 5-t-Butyl-2-oxo-[1,3]dioxol-4-ylmethyl Ester

(2S,4R)-5-Biphenyl-4-yl-2-methyl-2-(tetrahydropyran-2-yloxymethyl)-4-[(1-trityl-1H-1,2,3-triazole-4-carbonyl)amino]pentanoicacid (57 mg, 77 μmol) was combined with HOBt (31 mg, 230 μmol) and EDC(41 μL, 230 μmol) in DCM (5 mL) and stirred for 15 minutes. DMF (0.7 mL,10 mmol) was added and the resulting mixture was stirred for 15 minutes.4-t-Butyl-5-hydroxymethyl-1,3-dioxol-2-one (40 mg, 230 μmol) and4-methylmorpholine (34 μL, 0.31 mmol) were added and the mixture wasstirred at room temperature overnight. Water was added and the mixturewas extracted with EtOAc (20 mL), the organic layer was dried, and thesolvent evaporated. The reaction was monitored then quenched (1N HCl inwater with MeCN). 1.2M HCl in MeOH (10-20 volumes) was added and themixture was stirred for 2 hours, then purified by preparative HPLC toyield the title compound (1.6 mg). MS m/z [M+H]⁺ calc'd for C₃₀H₃₄N₄O₇,563.24; found 563.

1B:(2S,4R)-5-Biphenyl-4-yl-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid 2,2,3,3,3,3-Pentafluoropropyl Ester

(2S,4R)-5-Biphenyl-4-yl-2-methyl-2-(tetrahydropyran-2-yloxymethyl)-4-[(1-trityl-1H-1,2,3-triazole-4-carbonyl)amino]pentanoicacid (57 mg, 77 μmol) was combined with HOBt (31 mg, 230 μmol) and EDC(41 μL, 230 μmol) in DCM (5 mL) and stirred for 15 minutes. DMF (0.7 mL,10 mmol) was added and the resulting mixture was stirred for 15 minutes.2,2,3,3,3-Pentafluoro-1-propanol (23.2 μL, 230 μmol) and4-methylmorpholine (34 μL, 0.31 mmol) were added and the mixture wasstirred at room temperature overnight. Water was added and the mixturewas extracted with EtOAc (20 mL), the organic layer was dried, and thesolvent evaporated. The reaction was monitored then quenched (1N HCl inwater with MeCN). 1.2M HCl in MeOH (10-20 volumes) was added and themixture was stirred for 2 hours, then purified by preparative HPLC toyield the title compound (1.2 mg). MS m/z [M+H]⁺ calc'd forC₂₅H₂₅F₅N₄O₄, 541.18; found 541.

1C:(2S,4R)-5-Biphenyl-4-yl-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)-amino]pentanoicAcid 2,2-Difluoropropyl Ester

(2S,4R)-5-Biphenyl-4-yl-2-methyl-2-(tetrahydropyran-2-yloxymethyl)-4-[(1-trityl-1H-1,2,3-triazole-4-carbonyl)amino]pentanoicacid (57 mg, 77 μmol) was combined with HOBt (31 mg, 230 μmol) and EDC(41 μL, 230 μmol) in DCM (5 mL) and stirred for 15 minutes. DMF (0.7 mL,10 mmol) was added and the resulting mixture was stirred for 15 minutes.2,2-Difluoropropanol (22.3 mg, 230 μmol) and 4-methylmorpholine (34 μL,0.31 mmol) were added and the mixture was stirred at room temperatureovernight. Water was added and the mixture was extracted with EtOAc (20mL), the organic layer was dried, and the solvent evaporated. Thereaction was monitored then quenched (1N HCl in water with MeCN). 1.2MHCl in MeOH (10-20 volumes) was added and the mixture was stirred for 2hours, then purified by preparative HPLC to yield the title compound(1.4 mg). MS m/z [M+H]⁺ calc'd for C₂₅H₂₈F₂N₄O₄, 487.21; found 487.

1D:(2S,4R)-2-Acetoxymethyl-5-biphenyl-4-yl-2-methyl-4-[(3H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

(2S,4R)-5-Biphenyl-4-yl-2-methyl-2-(tetrahydropyran-2-yloxymethyl)-4-[(1H-1,2,3-triazole-4-carbonyl)amino]pentanoicacid (126 mg, 255 μmol) was combined with 4 M HCl in dioxane (191 μL,765 μmol) in MeCN (0.7 mL, 10 mmol). The mixture was then concentratedunder reduced pressure and the residue was purified by reverse phasechromatography. DCM (1 mL, 20 mmol) and acetyl chloride (24 mg, 306μmol) were added, followed by DIPEA (133 μL, 765 μmol). The resultingmixture was stirred for 10 minutes. The solvent was evaporated and theresidue was dissolved in AcOH and purified by preparative HPLC to yieldthe title compound (5 mg). MS m/z [M+H]⁺ calc'd for C₂₄H₂₆N₄O₅, 451.19;found 451.

1E:(2S,4R)-5-Biphenyl-4-yl-2-isobutyryloxymethyl-2-methyl-4-[(3H-[2,3]triazole-4-carbonyl)amino]pentanoicAcid

(2S,4R)-5-Biphenyl-4-yl-2-methyl-2-(tetrahydropyran-2-yloxymethyl)-4-[(1H-1,2,3-triazole-4-carbonyl)amino]pentanoicacid (126 mg, 255 μmol) was combined with 4 M HCl in dioxane (191 μL,765 μmol) in MeCN (0.7 mL, 10 mmol). The mixture was then concentratedunder reduced pressure and the residue was purified by reverse phasechromatography. DCM (1 mL, 20 mmol) and isobutyryl chloride (32.6 mg,306 μmol) were added, followed by DIPEA (133 μL, 765 μmol). Theresulting mixture was stirred for 10 minutes. The solvent was evaporatedand the residue was dissolved in AcOH and purified by preparative HPLCto yield the title compound (5 mg). MS m/z [M+H]⁺ calc'd for C₂₆H₃₀N₄O₅,479.22; found 479.

1F:(2S,4R)-5-Biphenyl-4-yl-2-methyl-2-(3-methylbutyryloxymethyl)-4-[(3H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

(2S,4R)-5-Biphenyl-4-yl-2-methyl-2-(tetrahydropyran-2-yloxymethyl)-4-[(1H-1,2,3-triazole-4-carbonyl)amino]pentanoicacid (126 mg, 255 μmol) was combined with 4 M HCl in dioxane (191 μL,765 μmol) in MeCN (0.7 mL, 10 mmol). The mixture was then concentratedunder reduced pressure and the residue was purified by reverse phasechromatography. DCM (1 mL, 20 mmol) and isovaleryl chloride (39.9 mg,306 μmol) were added, followed by DIPEA (133 μL, 765 μmol). Theresulting mixture was stirred for 10 minutes. The solvent was evaporatedand the residue was dissolved in AcOH and purified by preparative HPLCto yield the title compound (3 mg). MS m/z [M+H]⁺ calc'd for C₂₇H₃₂N₄O₅,493.24; found 493.

1G:(2S,4R)-5-Biphenyl-4-yl-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid Hexyl Ester

(2S,4R)-5-Biphenyl-4-yl-2-methyl-2-(tetrahydropyran-2-yloxymethyl)-4-[(1H-1,2,3-triazole-4-carbonyl)amino]pentanoicacid (100 mg, 0.2 mmol) was combined with 1-hexanol (0.3 mL, 2 mmol) and4 M HCl in 1,4-dioxane (0.3 mL, 1 mmol). The mixture was stirred for 2hours at 60° C. The mixture was concentrated under reduced pressure andthe residue was purified by reverse phase chromatography to yield thetitle compound (51 mg). MS m/z [M+H]⁺ calc'd for C₂₈H₃₆N₄O₄, 493.27;found 493.

1H:(2S,4R)-5-Biphenyl-4-yl-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid Heptyl Ester

(2S,4R)-5-Biphenyl-4-yl-2-methyl-2-(tetrahydropyran-2-yloxymethyl)-4-[(1H-1,2,3-triazole-4-carbonyl)amino]pentanoicacid (100 mg, 0.2 mmol) was combined with 1-heptanol (0.3 mL, 2 mmol)and 4 M HCl in 1,4-dioxane (0.3 mL, 1 mmol). The mixture was stirred for2 hours at 60° C. The mixture was concentrated under reduced pressureand the residue was purified by reverse phase chromatography to yieldthe title compound (60 mg). MS m/z [M+H]⁺ calc'd for C₂₉H₃₈N₄O₄, 507.29;found 507.

1I:(2S,4R)-5-Biphenyl-4-yl-2-ethoxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

1H-[1,2,3]Triazole-4-carboxylic acid and(2S,4R)-4-amino-5-biphenyl-4-yl-2-ethoxymethyl-2-methylpentanoic acidwere reacted as described herein to yield the title compound (0.8 mg).MS m/z [M+H]⁺ calc'd for C₂₄H₂₈N₄O₄, 437.21; found 437.2.

1J:(2S,4R)-5-Biphenyl-4-yl-2-methoxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

3H-[1,2,3]Triazole-4-carboxylic acid (3.5 mg, 31 μmol) and HATU (12 mg,31 μmol) were combined in DMF (0.5 mL) and stirred for 5 minutes. Asolution of(2S,4R)-4-amino-5-biphenyl-4-yl-2-methoxymethyl-2-methylpentanoic acidethyl ester (11 mg, 31 μmol) and DIPEA (16 μL, 93 μmol) in DMF (0.5 mL)was added and the resulting mixture was stirred for 20 minutes thenconcentrated under reduced pressure.

The residue was combined with THF (0.6 mL) and NaOH (124 μL, 124 μmol)and stirred at 60° C. for 2 hours, then concentrated under reducedpressure. The residue was dissolved in AcOH and compounds was purifiedby preparative HPLC to yield the title compound (1 mg). MS m/z [M+H]⁺calc'd for C₂₃H₂₆N₄O₄, 423.20; found 423.2.

Example 2

It is understood that the compounds of Example 2 can exist in a tautomerform, and that both forms are covered by this example. For example,(2S,4R)-2-hydroxymethyl-5-(2′-methoxybiphenyl-4-yl)-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicacid is depicted in Example 2A but it is understood that this compoundcan exist in a tautomer form, for example, as(2S,4R)-2-hydroxymethyl-5-(2′-methoxybiphenyl-4-yl)-2-methyl-4-[(3H-[1,2,3]triazole-4-carbonyl)amino]pentanoicacid. The same is true for the compounds in Examples 2B-2S.

2A:(2S,4R)-2-Hydroxymethyl-5-(2′-methoxybiphenyl-4-yl)-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

(2S,4R)-5-(4-bromophenyl)-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicacid (38 mg, 92 μmol) was combined with 2-methoxyphenylboronic acid(28.1 mg, 185 μmol), sodium carbonate (29.4 mg, 277 μmol), water (0.2mL) and dioxane (1.5 mL). The reaction vessel was sealed, air wasremoved by vacuum, and the vessel was purged with nitrogen.Tetrakis(triphenylphosphine)palladium (0) (21.4 mg, 18 μmol) was quicklyadded and air was removed by vacuum. The mixture was heated at 90° C.for 45 minutes. The mixture was acidified to pH ˜3 and filtered, and thesolvate was concentrated. The residue was dissolved in AcOH (0.7 mL) andpurified by preparative HPLC to yield the title compound (12.5 mg). MSm/z [M+H]⁺ calc'd for C₂₃H₂₆N₄O₅, 439.19; found 439.2.

2B:(2S,4R)-5-(2′-Chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

(2S,4R)-5-(4-bromophenyl)-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicacid (38 mg, 92 μmol) was combined with 2-chlorophenylboronic acid (28.9mg, 185 μmol), sodium carbonate (29.4 mg, 277 μmol), water (0.2 mL) anddioxane (1.5 mL). The reaction vessel was sealed, air was removed byvacuum, and the vessel was purged with nitrogen.Tetrakis(triphenylphosphine)palladium (0) (21.4 mg, 18 μmol) was quicklyadded and air was removed by vacuum. The mixture was heated at 90° C.for 45 minutes. The mixture was acidified to pH ˜3 and filtered, and thesolvate was concentrated. The residue was dissolved in AcOH (0.7 mL) andpurified by preparative HPLC to yield the title compound (18.2 mg). MSm/z [M+H]⁺ calc'd for C₂₂H₂₃ClN₄O₄, 443.14; found 443.2.

2C:(2S,4R)-2-Hydroxymethyl-2-methyl-5-(2′-methylbiphenyl-4-yl)-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

(2S,4R)-5-(4-bromophenyl)-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicacid (38 mg, 92 μmol) was combined with 2-methylphenylboronic acid (25.1mg, 185 μmol), sodium carbonate (29.4 mg, 277 μmol), water (0.2 mL) anddioxane (1.5 mL). The reaction vessel was sealed, air was removed byvacuum, and the vessel was purged with nitrogen.Tetrakis(triphenylphosphine)palladium (0) (21.4 mg, 18 μmol) was quicklyadded and air was removed by vacuum. The mixture was heated at 90° C.for 45 minutes. The mixture was acidified to pH ˜3 and filtered, and thesolvate was concentrated. The residue was dissolved in AcOH (0.7 mL) andpurified by preparative HPLC to yield the title compound (13.3 mg). MSm/z [M+H]⁺ calc'd for C₂₃H₂₆N₄O₄, 423.20; found 423.2.

2D:(2S,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

(2S,4R)-5-(4-bromophenyl)-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicacid (38 mg, 92 μmol) was combined with 3-chlorophenylboronic acid (28.9mg, 185 μmol), sodium carbonate (29.4 mg, 277 μmol), water (0.2 mL) anddioxane (1.5 mL). The reaction vessel was sealed, air was removed byvacuum, and the vessel was purged with nitrogen.Tetrakis(triphenylphosphine)palladium (0) (21.4 mg, 18 μmol) was quicklyadded and air was removed by vacuum. The mixture was heated at 90° C.for 45 minutes. The mixture was acidified to pH ˜3 and filtered, and thesolvate was concentrated. The residue was dissolved in AcOH (0.7 mL) andpurified by preparative HPLC to yield the title compound (8.1 mg). MSm/z [M+H]⁺ calc'd for C₂₂H₂₃ClN₄O₄, 443.14; found 443.2.

2E:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

(2S,4R)-5-(4-bromophenyl)-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicacid (38 mg, 92 μmol) was combined with 5-chloro-2-fluorophenylboronicacid (32.2 mg, 185 μmol), sodium carbonate (29.4 mg, 277 μmol), water(0.2 mL) and dioxane (1.5 mL). The reaction vessel was sealed, air wasremoved by vacuum, and the vessel was purged with nitrogen.Tetrakis(triphenylphosphine)palladium (0) (21.4 mg, 18 μmol) was quicklyadded and air was removed by vacuum. The mixture was heated at 90° C.for 45 minutes. The mixture was acidified to pH ˜3 and filtered, and thesolvate was concentrated. The residue was dissolved in AcOH (0.7 mL) andpurified by preparative HPLC to yield the title compound (2 mg). MS m/z[M+H]⁺ calc'd for C₂₂H₂₂ClFN₄O₄, 461.13; found 461.2.

2F:(2S,4R)-5-(2′,5′-Dichlorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

(2S,4R)-5-(4-bromophenyl)-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicacid (38 mg, 92 μmol) was combined with 2,5-dichlorophenylboronic acid(35.3 mg, 185 μmol), sodium carbonate (29.4 mg, 277 μmol), water (0.2mL) and dioxane (1.5 mL). The reaction vessel was sealed, air wasremoved by vacuum, and the vessel was purged with nitrogen.Tetrakis(triphenylphosphine)palladium (0) (21.4 mg, 18 μmol) was quicklyadded and air was removed by vacuum. The mixture was heated at 90° C.for 45 minutes. The mixture was acidified to pH ˜3 and filtered, and thesolvate was concentrated. The residue was dissolved in AcOH (0.7 mL) andpurified by preparative HPLC to yield the title compound (4.6 mg). MSm/z [M+H]⁺ calc'd for C₂₂H₂₂Cl₂N₄O₄, 477.10; found 478.2.

2G:(2S,4R)-5-(5′-Chloro-2′-methylbiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

(2S,4R)-5-(4-bromophenyl)-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicacid (38 mg, 92 μmol) was combined with 5-chloro-2-methylphenylboronicacid (31.4 mg, 185 μmol), sodium carbonate (29.4 mg, 277 μmol), water(0.2 mL) and dioxane (1.5 mL). The reaction vessel was sealed, air wasremoved by vacuum, and the vessel was purged with nitrogen.Tetrakis(triphenylphosphine)palladium (0) (21.4 mg, 18 μmol) was quicklyadded and air was removed by vacuum. The mixture was heated at 90° C.for 45 minutes. The mixture was acidified to pH ˜3 and filtered, and thesolvate was concentrated. The residue was dissolved in AcOH (0.7 mL) andpurified by preparative HPLC to yield the title compound (12.1 mg). MSm/z [M+H]⁺ calc'd for C₂₃H₂₅ClN₄O₄, 457.16; found 457.2.

2H:(2S,4R)-5-(3′-Cyanobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

(2S,4R)-5-(4-Bromophenyl)-4-t-butoxycarbonylamino-2-hydroxymethyl-2-methylpentanoicacid (40 mg, 96 μmol) was combined with 3-cyanophenylboronic acid (14mg, 96 μmol), sodium carbonate (10.2 mg, 96 μmol), water (0.5 mL) anddioxane (2 mL). The reaction vessel was sealed, air was removed byvacuum, and the vessel was purged with nitrogen.Tetrakis(triphenylphosphine)palladium (0) (11 mg, 9.6 μmol) was quicklyadded, air was removed by vacuum, and the vessel was purged withnitrogen. The mixture was heated at 90° C. for 45 minutes. The mixturewas filtered and the solvate was concentrated. The residue was dissolvedin AcOH and purified by reverse phase chromatography to yield Compound 1(30 mg).

Compound 1 (30 mg, 68 μmol) was combined with MeCN (1 mL) and 4M HCl in1,4-dioxane (103 μL, 410 μmol) and stirred for 10 minutes. The mixturewas then concentrated under reduced pressure to yield Compound 2 (27mg).

3H-[1,2,3]triazole-4-carboxylic acid (9.1 mg, 80 μmol) was combined withHATU (30 mg, 80 μmol) in DMF (0.5 mL) and stirred for 5 minutes.Compound 2 (27 mg, 80 μmol) was added, followed by DIPEA (42 μL, 240μmol), and the resulting mixture was stirred for 30 minutes. The solventwas evaporated and the residue was dissolved in AcOH and purified byreverse phase to yield the title compound (3 mg). MS m/z [M+H]⁺ calc'dfor C₂₃H₂₃N₅O₄, 434.18; found 434.

2I:(2S,4R)-2-Hydroxymethyl-2-methyl-5-(3′-methylbiphenyl-4-yl)-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

(2S,4R)-5-(4-bromophenyl)-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicacid (38 mg, 92 μmol) was combined with 3-methylphenylboronic acid (25.1mg, 185 μmol), sodium carbonate (29.4 mg, 277 μmol), water (0.2 mL) anddioxane (1.5 mL). The reaction vessel was sealed, air was removed byvacuum, and the vessel was purged with nitrogen.Tetrakis(triphenylphosphine)palladium (0) (21.4 mg, 18 μmol) was quicklyadded and air was removed by vacuum. The mixture was heated at 90° C.for 45 minutes. The mixture was acidified to pH ˜3 and filtered, and thesolvate was concentrated. The residue was dissolved in AcOH (0.7 mL) andpurified by preparative HPLC to yield the title compound (10 mg).

2J:(2S,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-methoxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

3H-[1,2,3]Triazole-4-carboxylic acid (3.5 mg, 31 μmol) and HATU (12 mg,31 μmol) were combined in DMF (0.5 mL) and stirred for 5 minutes. Asolution of(2S,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-methoxymethyl-2-methylpentanoicacid ethyl ester (12 mg, 31 μmol) and DIPEA (16 μL, 93 μmol) in DMF (0.5mL) was added and the resulting mixture was stirred for 20 minutes thenconcentrated under reduced pressure.

The residue was combined with THF (0.6 mL) and NaOH (124 μL, 124 μmol)and stirred at 60° C. for 2 hours, then concentrated under reducedpressure. The residue was dissolved in AcOH and purified by reversephase chromatography to yield the title compound (4.0 mg). MS m/z [M+H]⁺calc'd for C₂₃H₂₅ClN₄O₄, 457.16; found 457.2.

2K:(2S,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-ethoxymethyl-2-methyl-4-[(3H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

Into a vial was added(2S,4R)-4-t-butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid ethyl ester (400 mg, 840 μmol), tetrabutylammonium hydrogen sulfate(57 mg, 168 μmol), DCM (1 mL) and NaOH (588 μL, 5.9 mmol), followed bydiethylsulfate (518 mg, 3.4 mmol). The reaction vessel was capped andstirred vigorously overnight. The mixture was extracted with DCM andwater, purified (normal phase chromatography 0-60% EtOAc:hexanes), thenconcentrated under reduced pressure to yield Compound 1 (180 mg).

Compound 1 (87 mg, 173 μmol) in MeCN (1 mL) was combined with 4N HCl indioxane (0.3 mL). The mixture was stirred for 10 minutes thenconcentrated under reduced pressure to yield Compound 2.

Compound 2 (33.7 mg, 83 μmol) was combined with HATU (38.0 mg, 100μmol), 3H-[1,2,3]triazole-4-carboxylic acid (12.3 mg, 108 μmol) in DMF(0.5 mL). DIPEA (43.7 μL, 250 μmol) was added and the mixture wasstirred for 2 hours. EtOAc was added, followed by a saturated aqueousNH₄Cl solution. The mixture was stirred for 10 minutes then concentratedunder reduced pressure to yield Compound 3.

Compound 3 (40.7 mg, 82 μmol) was combined with THF (0.6 mL) and NaOH(326 μL, 326 μmol) and a few drop of MeOH. The resulting mixture wasstirred at 60° C. for 2 hours, then concentrated under reduced pressure.The residue was dissolved in AcOH and purified by reverse phase to yieldthe title compound (12 mg). MS m/z [M+H]⁺ calc'd for C₂₄H₂₇ClN₄O₄,471.17; found 471.2.

2L:(2S,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-(2-hydroxyethoxymethyl)-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

Into a vial was added(2S,4R)-4-t-butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid ethyl ester (400 mg, 840 μmol), tetrabutylammonium hydrogen sulfate(57 mg, 168 μmol), DCM (1 mL) and NaOH (588 μL, 5.9 mmol), followed by[1,3,2]dioxathiolane 2,2-dioxide (417 mg, 3.4 mmol). The reaction vesselwas capped and stirred vigorously overnight. The mixture was extractedwith DCM and water, purified (normal phase chromatography 0-60%EtOAc:hexanes), then concentrated under reduced pressure to yieldCompound 1 (90 mg).

Compound 1 (90 mg, 173 μmol) in MeCN (1 mL) was combined with 4N HCl indioxane (0.3 mL). The mixture was stirred for 10 minutes thenconcentrated under reduced pressure to yield Compound 2.

Compound 2 (35 mg, 83 μmol) was combined with HATU (38 mg, 100 μmol),1H-[1,2,3]triazole-4-carboxylic acid (12.3 mg, 108 μmol) in DMF (0.5mL). DIPEA (43.7 μL, 250 μmol) was added and the mixture was stirred for2 hours. EtOAc was added, followed by a saturated aqueous NH₄Clsolution. The mixture was stirred for 10 minutes then concentrated underreduced pressure to yield Compound 3.

Compound 3 (42 mg, 82 μmol) was combined with THF (0.6 mL) and NaOH (326μL, 326 μmol) and a few drop of MeOH. The resulting mixture was stirredat 60° C. for 2 hours, then concentrated under reduced pressure. Theresidue was dissolved in AcOH and purified by reverse phase to yield thetitle compound (11 mg). MS m/z [M+H]⁺ calc'd for C₂₄H₂₇ClN₄O₅, 487.17;found 487.2.

2M:(2S,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-(3-hydroxypropoxymethyl)-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

Into a vial was added(2S,4R)-4-t-butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid ethyl ester (67 mg, 140 μmol), tetrabutylammonium hydrogen sulfate(9.5 mg, 28 μmol), DCM (1 mL) and NaOH (98 μL, 982 μmol), followed by1,3-propanediol cyclic sulfate (78 mg, 561 μmol). The mixture wasstirred overnight then extracted with DCM and purified (normal phasechromatography (0-100% EtOAc:hexanes) to yield Compound 1 (7 mg).

Compound 1 (26.3 mg, 49 μmol) in MeCN (0.3 mL) was combined with 4N HClin dioxane (0.3 mL). The mixture was stirred for 10 minutes thenconcentrated under reduced pressure to yield Compound 2.

Compound 2 (18 mg, 47 μmol) was dissolved in DMF (0.3 mL) and1H-1,2,3-triazole-4-carboxylic acid (5.3 mg, 47 μmol). HATU (18 mg, 47μmol) was added, followed by DIPEA (25 μL, 141 μmol). The mixture wasstirred for 30 minutes then concentrated under reduced pressure to yieldCompound 3, which was used without further purification.

Compound 3 (23 mg, 47 μmol) was dissolved in THF and NaOH (188 μL, 188mol) was added and the mixture was stirred at 60° C. overnight. theresidue was dissolved in AcOH and purified by preparative HPLC to yieldthe title compound (1.2 mg). MS m/z [M+H]⁺ calc'd for C₂₅H₂₉ClN₄O₅,501.18; found 502.2.

2N:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-methoxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

Into a vial was added(2S,4R)-4-t-butoxycarbonylamino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid ethyl ester (82 mg, 166 μmol), tetrabutylammonium hydrogen sulfate(11 mg, 33 μmol), DCM (1 mL) and NaOH (116 μL, 1.2 mmol), followed bydimethyl sulfate (84 mg, 664 mmol). The reaction vessel was capped andstirred vigorously overnight. The mixture was extracted with DCM andconcentrated under reduced pressure. The residue was dissolved in AcOHand purified by reverse phase chromatography (30-100% MeCN in water) toyield Compound 1 (30 mg).

Compound 1 (84 mg, 166 μmol) in MeCN (1 mL) was combined with 4N HCl indioxane (0.3 mL). The mixture was stirred for 10 minutes thenconcentrated under reduced pressure to yield Compound 2.

3H-[1,2,3]triazole-4-carboxylic acid (21 mg, 183 μmol) was combined withHATU (69 mg, 183 μmol), in DMF (0.5 mL), Compound 2 (68 mg, 166 μmol),and DIPEA (87 μL, 498 μmol). The resulting mixture was stirred for 20minutes then concentrated under reduced pressure. The residue waspurified (normal phase chromatography 0-80% EtOAc:hexanes) to yieldCompound 3.

Compound 3 (65 mg, 129 μmol) was combined with THF (0.6 mL) and NaOH(516 μL, 516 μmol). The resulting mixture was stirred at 60° C. for 2hours. A small amount of NaOH and MeOH was added and the mixture wasstirred overnight. The mixture was acidified with concentrated HCl topH-4, then concentrated under reduced pressure. The residue wasdissolved in AcOH and purified by preparative HPLC to yield the titlecompound (35 mg). MS m/z [M+H]⁺ calc'd for C₂₃H₂₄ClFN₄O₄, 475.15; found475.2.

2O:(2S,4R)-5-(5′-Chloro-2′-fluoro-biphenyl-4-yl)-2-ethoxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

Into a vial was added(2S,4R)-4-t-butoxycarbonylamino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid ethyl ester (415 mg, 840 μmol), tetrabutylammonium hydrogen sulfate(57 mg, 168 μmol), DCM (1 mL) and NaOH (588 μL, 5.9 mmol), followed bydiethylsulfate (518 mg, 3.4 mmol). The reaction vessel was capped andstirred vigorously overnight. The mixture was extracted with DCM andwater, purified (normal phase chromatography 0-60% EtOAc:hexanes), thenconcentrated under reduced pressure to yield Compound 1 (110 mg).

Compound 1 (90 mg, 173 mol) in MeCN (1 mL) was combined with 4N HCl indioxane (0.3 mL). The mixture was stirred for 10 minutes thenconcentrated under reduced pressure to yield Compound 2.

Compound 2 (35.2 mg, 83 μmol) was combined with HATU (38.0 mg, 100μmol), 3H-[1,2,3]triazole-4-carboxylic acid (12.3 mg, 108 μmol) in DMF(0.5 mL). DIPEA (43.7 μL, 250 μmol) was added and the mixture wasstirred for 2 hours. EtOAc was added, followed by a saturated aqueousNH₄Cl solution. The mixture was stirred for 10 minutes then concentratedunder reduced pressure to yield Compound 3.

Compound 3 (42.2 mg, 82 μmol) was combined with THF (0.6 mL) and NaOH(326 μL, 326 μmol) and a few drops of MeOH. The resulting mixture wasstirred at 60° C. for 2 hours, then concentrated under reduced pressure.The residue was dissolved in AcOH and purified by reverse phase to yieldthe title compound (23 mg). MS m/z [M+H]⁺ calc'd for C₂₄H₂₆ClFN₄O₄,489.16; found 489.2.

2P:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-(3-hydroxypropoxymethyl)-2-methyl-4-[(3H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

The title compounds was also prepared (4 mg). MS m/z [M+H]⁺ calc'd forC₂₅H₂₈ClFN₄O₅, 519.17; found 519.

2Q.(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-methyl-2-pentyloxymethyl-4-[(3H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

The title compounds was also prepared (6 mg). MS m/z [M+H]⁺ calc'd forC₂₇H₃₂ClFN₄O₄, 531.21; found 531.

2R:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-isopropoxymethyl-2-methyl-4-[(3H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

The title compounds was also prepared (7 mg). MS m/z [M+H]⁺ calc'd forC₂₅H₂₈ClFN₄O₄, 503.18; found 503.

2S:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-(2-hydroxyethoxymethyl)-2-methyl-4-[(3H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

The title compounds was also prepared (4 mg). MS m/z [M+H]⁺ calc'd forC₂₄H₂₄FN₇O₂, 462.20; found 462.2.

Example 3

It is understood that the compounds of Example 3 can exist in a tautomerform, and that both forms are covered by this example. For example,(2S,4R)-5-(3′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(3H-[1,2,3]triazole-4-carbonyl)amino]pentanoicacid is depicted in Example 3A but it is understood that this compoundcan exist in a tautomer form, for example, as(2S,4R)-5-(3′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(1H-[1,2,3]triazole-4-carbonyl)amino]pentanoicacid. The same is true for the compound in Example 3B.

3A:(2S,4R)-5-(3′-Fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(3H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

1,2,3-Triazole-4-carboxylic acid (27.3 mg, 241 μmol) was combined withEDC (42.7 μL, 241 μmol), 4-methylmorpholine (1 eq.) and HOBt (32.6 mg,241 μmol) in DMF (0.2 mL). The resulting mixture was stirred for 5minutes at room temperature. A solution(2S,4R)-4-amino-5-(3′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (80 mg, 240 μmol) and 4-methylmorpholine (53.1 μL, 483 μmol) in DMF(0.3 mL) was added, and the resulting mixture was stirred for 15minutes. The reaction was quenched with ACOH and the product waspurified by preparative HPLC and lyophilized to yield the title compound(30 mg). MS m/z [M+H]⁺ calc'd for C₂₂H₂₃FN₄O₄, 427.17; found 427.2.

3B:(2S,4R)-5-(2′-Fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(3H-[1,2,3]triazole-4-carbonyl)amino]pentanoicAcid

1,2,3-Triazole-4-carboxylic acid (30 mg, 260 μmol) was combined withDIPEA (92.4 μL, 531 μmol) and HATU (101 mg, 265 μmol) in DMF (0.2 mL).The resulting mixture was stirred for 5 minutes at room temperature. Asolution(2S,4R)-4-t-butoxycarbonylamino-5-(2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (114 mg, 265 μmol) and DIPEA (3 eq.) in DMF (0.2 mL) was added, andthe resulting mixture was stirred for 15 minutes. The reaction wasquenched with ACOH and the product was purified by preparative HPLC andlyophilized to yield the title compound (16 mg). MS m/z [M+H]⁺ calc'dfor C₂₂H₂₃FN₄O₄, 427.17; found 427.2.

Example 4 4A:(2S,4R)-5-(2′-Fluorobiphenyl-4-yl)-2-hydroxymethyl-4-[(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)amino]-2-methylpentanoicAcid

1-Hydroxy-1H-1,2,3-triazole-4-carboxylic acid (15 mg, 116 μmol) wascombined with DIPEA (40.5 μL, 232 μmol) and HATU (44.2 mg, 116 μmol) inDMF (0.2 mL). The resulting mixture was stirred for 5 minutes at roomtemperature. A solution(2S,4R)-4-amino-5-(2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (38.5 mg, 166 μmol) and DIPEA (3 eq.) in DMF (0.2 mL) was added,and the resulting mixture was stirred for 15 minutes. The reaction wasquenched with ACOH and the product was purified by preparative HPLC andlyophilized to yield the title compound (8 mg). MS m/z [M+H]⁺ calc'd forC₂₂H₂₃FN₄O₅, 443.17; found 443.2.

4B:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-4-[(1-methoxy-1H-[1,2,3]triazole-4-carbonyl)amino]-2-methylpentanoicAcid

1-Methoxy-1H-[1,2,3]triazole-4-carboxylic acid (4.3 mg, 30 μmol) andHATU (11.4 mg, 30 μmol) were combined in DMF (1 mL) and stirred at roomtemperature for 15 minutes.(2S,4R)-4-amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (10 mg, 27 μmol) and DIPEA (14 μL, 82 μmol) were added, and theresulting mixture was stirred for 15 minutes at room temperature. Thesolvent was removed in vacuo and the residue was purified by preparativeHPLC to yield the title compound (1.1 mg). MS m/z [M+H]⁺ calc'd forC₂₃H₂₄ClFN₄O₅, 491.14; found 491.2.

4C:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-4-[(1-ethoxy-1H-[1,2,3]triazole-4-carbonyl)-amino]-2-hydroxymethyl-2-methylpentanoicAcid

1-Ethoxy-1H-[1,2,3]triazole-4-carboxylic acid (4.7 mg, 30 μmol) and HATU(11.4 mg, 30 μmol) were combined in DMF (1 mL) and stirred at roomtemperature for 15 minutes.(2S,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (10 mg, 27 μmol) and DIPEA (14 μL, 82 μmol) were added, and theresulting mixture was stirred for 15 minutes at room temperature. Thesolvent was removed in vacuo and the residue was purified by preparativeHPLC to yield the title compound (2 mg). MS m/z [M+H]⁺ calc'd forC₂₄H₂₆ClFN₄O₅, 505.16; found 505.1.

Example 5 5A:(2S,4R)-5-(2′-Fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(2-oxo-2,3-dihydrooxazole-4-carbonyl)amino]pentanoicAcid

THF (1 mL, 10 mmol), ethyl 2-oxo-2,3-dihydrooxazole-4-carboxylate (12mg, 76.4 μmol), and 1 M NaOH in water (229 μL, 229 μmol) were combinedand stirred until completion. The mixture was acidified to pH˜5 with 1NHCl, the solvent was evaporated in vacuo, and the product was azeotropedin toluene and dried in vacuo. To this was added a solution of DIPEA(26.6 μL, 153 μmol) and HATU (29.0 mg, 76.4 μmol) in DMF (0.2 mL), andthe resulting mixture was stirred for 5 minutes at room temperature.(2S,4R)-4-Amino-5-(2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (25.3 mg, 76.4 μmol) was added and the resulting mixture wasstirred for 15 minutes. The reaction was quenched with EtOAc andsaturated NH₄Cl. The product was extracted and dried. AcOH was added andthe product was purified by preparative HPLC to yield the title compound(2.5 mg). MS m/z [M+H]⁺ calc'd for C₂₃H₂₃FN₂O₆, 443.15; found 443.2.

5B:(2S,4R)-5-(2′-Fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(2-oxo-2,3-dihydrooxazole-5-carbonyl)amino]pentanoicAcid

THF (1 mL, 10 mmol), ethyl 2-oxo-2,3-dihydrooxazole-5-carboxylate (12mg, 76.4 μmol), and 1 M NaOH in water (229 μL, 229 μmol) were combinedand stirred until completion. The mixture was acidified to pH˜5 with 1NHCl, the solvent was evaporated in vacuo, and the product was azeotropedin toluene and dried in vacuo. To this was added a solution of DIPEA(26.6 μL, 153 μmol) and HATU (29.0 mg, 76.4 μmol) in DMF (0.2 mL), andthe resulting mixture was stirred for 5 minutes at room temperature.(2S,4R)-4-Amino-5-(2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (25.3 mg, 76.4 μmol) was added and the resulting mixture wasstirred for 15 minutes. The reaction was quenched with EtOAc andsaturated NH₄Cl. The product was extracted and dried. AcOH was added andthe product was purified by preparative HPLC to yield the title compound(5 mg). MS m/z [M+H]⁺ calc'd for C₂₃H₂₃FN₂O₆, 443.15; found 443.2.

5C:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(2-oxo-2,3-dihydro-oxazole-4-carbonyl)amino]pentanoicAcid

2-Oxo-2,3-dihydrooxazole-4-carboxylic acid (7 mg, 55 μmol) and HATU(20.8 mg, 55 μmol) were combined in DMF (0.2 mL) and allowed to stand atroom temperature for 10 minutes.(2S,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (20 mg, 55 μmol) in DMF and DIPEA (28.6 μL, 164 μmol) were added,and the resulting mixture was stirred for 20 minutes at roomtemperature. The mixture was concentrated under reduced pressure and theresidue was dissolved in AcOH purified by preparative HPLC to yield thetitle compound (0.6 mg). MS m/z [M+H]⁺ calc'd for C₂₃H₂₂ClFN₂O₆, 477.12;found 477.2.

5D:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(2-oxo-2,3-dihydro-oxazole-5-carbonyl)amino]pentanoicAcid

2-Oxo-2,3-dihydrooxazole-5-carboxylic acid (7.1 mg, 55 μmol) and HATU(21 mg, 55 μmol) were combined in DMF (0.3 mL) and stirred at roomtemperature for 5 minutes.(2S,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (20 mg, 55 μmol) in DMF (0.5 mL) and DIPEA (29 μL, 164 μmol) wereadded, and the resulting mixture was stirred for 10 minutes at roomtemperature. The mixture was concentrated under reduced pressure and theresidue was dissolved in AcOH purified by preparative HPLC to yield thetitle compound (3 mg). MS m/z [M+H]±calc'd for C₂₃H₂₂ClFN₂O₆, 477.12;found 477.

Example 6 6A:(2S,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxymethyl-4-[(3-methoxyisoxazole-5-carbonyl)amino]-2-methylpentanoicAcid

3-Methoxyisoxazole-5-carboxylic acid (9 mg, 32 μmol) was combined withHATU (12 mg, 32 μmol) and DMF (0.2 mL) and the resulting mixture wasstirred for 5 minutes. DIPEA (17 μL, 96 μmol) and(2S,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (79 mg, 38 μmol) pre-dissolved in DMF were added and the resultingmixture was stirred for 15 minutes then concentrated. The residue wasdissolved in AcOH and purified by preparative HPLC to yield the titlecompound (2.2 mg). MS m/z [M+H]⁺ calc'd for C₂₄H₂₅C₁N₂O₆, 473.14; found473.2.

6B:(2S,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-[(3-methoxyisoxazole-5-carbonyl)amino]-2-methoxymethyl-2-methylpentanoicAcid

3-Methoxyisoxazole-5-carboxylic acid (4.4 mg, 31 μmol) and HATU (12 mg,31 μmol) were combined in DMF (0.5 mL) and stirred for 5 minutes. Asolution of(2S,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-methoxymethyl-2-methylpentanoicacid ethyl ester (12 mg, 31 μmol) and DIPEA (16 μL, 93 μmol) in DMF (0.5mL) was added and the resulting mixture was stirred for 20 minutes thenconcentrated under reduced pressure.

The residue was combined with THF (0.6 mL) and NaOH (124 μL, 124 μmol)and stirred at 60° C. for 2 hours, then concentrated under reducedpressure. The residue was dissolved in AcOH and compounds was purifiedby preparative HPLC to yield the title compound (1 mg). MS m/z [M+H]⁺calc'd for C₂₅H₂₇ClN₂O₆, 487.16; found 487.2.

6C:(2S,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-ethoxymethyl-4-[(3-methoxyisoxazole-5-carbonyl)amino]-2-methylpentanoicAcid

3-Methoxyisoxazole-5-carboxylic acid and(2S,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-ethoxymethyl-2-methylpentanoicacid were reacted as described herein to yield the title compound (2mg). MS m/z [M+H]⁺ calc'd for C₂₆H₂₉ClN₂O₆, 501.17; found 501.2.

6D:(2S,4R)-5-Biphenyl-4-yl-4-[(3-hydroxyisoxazole-5-carbonyl)amino]-2-hydroxymethyl-2-methylpentanoicAcid

3-Hydroxy-isoxazole-5-carboxylic acid (10.6 mg, 82 μmol), EDC (14.5 μL,82 μmol), and HOBt (11.1 mg, 82 μmol) were combined in DMF (0.2 mL) andstirred for 5 minutes.(2S,4R)-4-Amino-5-biphenyl-4-yl-2-hydroxymethyl-2-methylpentanoic acid(26 mg, 82 μmol) was added and the resulting mixture was stirred for 18hours. The reaction was quenched with AcOH and the product was purifiedby preparative HPLC then lyophilized to yield the title compound as aTFA salt (7 mg). MS m/z [M+H]⁺ calc'd for C₂₃H₂₄N₂O₆, 425.16; found425.4.

6E:(2S,4R)-5-Biphenyl-4-yl-[(3-hydroxyisoxazole-5-carbonyl)amino]-2-methoxymethyl-2-methylpentanoicAcid

3-Hydroxy-isoxazole-5-carboxylic acid and(2S,4R)-4-amino-5-biphenyl-4-yl-2-methoxymethyl-2-methylpentanoic acidethyl ester were reacted as described herein to yield the title compound(2.4 mg). MS m/z [M+H]⁺ calc'd for C₂₄H₂₆N₂O₆, 439.18; found 439.2.

6F:(2S,4R)-5-Biphenyl-4-yl-4-[(3-methoxyisoxazole-5-carbonyl)amino]-2-methoxymethyl-2-methylpentanoicAcid

3-Methoxyisoxazole-5-carboxylic acid (4.4 mg, 31 μmol) and HATU (12 mg,31 μmol) were combined in DMF (0.5 mL) and stirred for 5 minutes. Asolution of(2S,4R)-4-amino-5-biphenyl-4-yl-2-methoxymethyl-2-methylpentanoic acidethyl ester (11 mg, 31 μmol) and DIPEA (16 μL, 93 μmol) in DMF (0.5 mL)was added and the resulting mixture was stirred for 20 minutes thenconcentrated under reduced pressure.

The residue was combined with THF (0.6 mL) and NaOH (124 μL, 124 μmol)and stirred at 60° C. for 2 hours, then concentrated under reducedpressure. The residue was dissolved in AcOH and compounds was purifiedby preparative HPLC to yield the title compound (1 mg). MS m/z [M+H]⁺calc'd for C₂₅H₂₈N₂O₆, 453.19; found 453.

6G:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-4-[(3-methoxyisoxazole-5-carbonyl)amino]-2-methylpentanoicAcid

3-Methoxyisoxazole-5-carboxylic acid (8 mg, 55 μmol) and HATU (20.8 mg,55 μmol) were combined in DMF (0.2 mL) and allowed to stand at roomtemperature for 10 minutes.(2S,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (20 mg, 55 μmol) in DMF and DIPEA (28.6 μL, 164 μmol) were added,and the resulting mixture was stirred for 20 minutes at roomtemperature. The mixture was concentrated under reduced pressure and theresidue was dissolved in AcOH purified by preparative HPLC to yield thetitle compound (5.4 mg). MS m/z [M+H]⁺ calc'd for C₂₄H₂₄ClFN₂O₆, 491.13;found 491.2.

6H:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-4-[(3-ethylisoxazole-5-carbonyl)amino]-2-hydroxymethyl-2-methylpentanoicAcid

3-Ethylisoxazole-5-carboxylic acid (8 mg, 55 μmol) and HATU (20.8 mg, 55μmol) were combined in DMF (0.2 mL) and allowed to stand at roomtemperature for 10 minutes.(2S,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (20 mg, 55 μmol) in DMF and DIPEA (28.6 μL, 164 μmol) were added,and the resulting mixture was stirred for 20 minutes at roomtemperature. The mixture was concentrated under reduced pressure and theresidue was dissolved in AcOH purified by preparative HPLC to yield thetitle compound (3.6 mg). MS m/z [M+H]⁺ calc'd for C₂₅H₂₆ClFN₂O₅, 489.15;found 490.2.

6I:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-4-[(3-isobutylisoxazole-5-carbonyl)amino]-2-methylpentanoicAcid

3-Isobutylisoxazole-5-carboxylic acid (9 mg, 55 μmol) and HATU (20.8 mg,55 μmol) were combined in DMF (0.2 mL) and allowed to stand at roomtemperature for 10 minutes.(2S,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (20 mg, 55 μmol) in DMF and DIPEA (28.6 μL, 164 μmol) were added,and the resulting mixture was stirred for 20 minutes at roomtemperature. The mixture was concentrated under reduced pressure and theresidue was dissolved in AcOH purified by preparative HPLC to yield thetitle compound (0.3 mg). MS m/z [M+H]⁺ calc'd for C₂₇H₃₀ClFN₂O₅, 517.18;found 517.2.

6J:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-[(3-propylisoxazole-5-carbonyl)amino]pentanoicAcid

3-Propylisoxazole-5-carboxylic acid (9 mg, 55 μmol) and HATU (20.8 mg,55 μmol) were combined in DMF (0.2 mL) and allowed to stand at roomtemperature for 10 minutes.(2S,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (20 mg, 55 μmol) in DMF and DIPEA (28.6 μL, 164 μmol) were added,and the resulting mixture was stirred for 20 minutes at roomtemperature. The mixture was concentrated under reduced pressure and theresidue was dissolved in AcOH purified by preparative HPLC to yield thetitle compound (0.5 mg). MS m/z [M+H]⁺ calc'd for C₂₆H₂₈ClFN₂O₅, 503.17;found 504.2.

6K:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-4-[(3-hydroxyisoxazole-5-carbonyl)amino]-2-methoxymethyl-2-methylpentanoicAcid

3-Hydroxy-isoxazole-5-carboxylic acid (4 μg, 0.03 μmol) and HATU (11 μg,0.03 μmol) were combined with(2S,4R)-4-amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-methoxymethyl-2-methylpentanoicacid ethyl ester (10 μg, 0.03 μmol) in DMF (0.5 mL) and stirred for 5minutes. DIPEA (0.01 μL, 0.07 μmol) was added, and the resulting mixturewas stirred 20 min and evaporated to yield crude Compound 1, which wasused directly in the next step.

Compound 1 (10 mg) in THF (1 mL) was combined with 1N NaOH (0.3 mL) andthe resulting mixture was stirred at 60° C. for 3 hours. AcOH was addedand the product was purified (reverse phase) to yield the title compound(1 mg). MS m/z [M+H]⁺ calc'd for C₂₄H₂₄ClFN₂O₆, 491.13; found 491.

6L:(2S,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-(2-hydroxyethoxymethyl)-4-[(3-hydroxyisoxazole-5-carbonyl)amino]-2-methylpentanoicAcid

(2S,4R)-4-t-Butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid ethyl ester (415 mg, 840 μmol) and tetrabutylammonium hydrogensulfate (57 mg, 168 μmol) were combined with DCM (1 mL) and NaOH (588μL, 5.9 mmol). [1,3,2]Dioxathiolane 2,2-dioxide (424 mg, 3.4 mmol) wasadded and the reaction vessel was sealed and stirred vigorouslyovernight. The mixture was extracted with DCM and water then purified(normal phase chromatography 0-60% EtOAc to hexanes) to yield Compound 1(90 mg).

Compound 1 (90 mg, 173 μmol) was combined with MeCN (1 mL) and 4N HCl indioxane (0.3 mL) and stirred for 10 minutes, then concentrated underreduced pressure to yield Compound (2).

Compound 2 (35 mg, 83 μmol), HATU (38.0 mg, 100 μmol),3-Hydroxy-isoxazole-5-carboxylic acid (12.3 mg, 108 μmol) and DMF (0.5mL) were combined, followed by DIPEA (43.7 μL, 250 μmol). The resultingmixture was stirred for 2 hours. EtOAc was added, then saturated aqueousNH₄Cl. The mixture was then concentrated under reduced pressure. Theresidue was combined with THF (0.6 mL) and NaOH (326 μL, 326 μmol) witha few drop of MeOH, and stirred at 60° C. for 2 hours. The mixture wasthen concentrated under reduced pressure. The residue was dissolved inAcOH and purified by preparative HPLC to yield the title compound (8mg). MS m/z [M+H]⁺ calc'd for C₂₅H₂₇ClN₂O₇, 503.15; found 503.

6M:(2S,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-ethoxymethyl-4-[(3-hydroxyisoxazole-5-carbonyl)amino]-2-methylpentanoicAcid

(2S,4R)-4-t-Butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid ethyl ester (415 mg, 840 μmol) and tetrabutylammonium hydrogensulfate (57 mg, 168 μmol) were combined with DCM (1 mL) and NaOH (588μL, 5.9 mmol). Diethylsulfate (518 mg, 3.4 mmol) was added and thereaction vessel was sealed and stirred vigorously overnight. The mixturewas extracted with DCM and water then purified (normal phasechromatography 0-60% EtOAc to hexanes) to yield Compound 1 (180 mg).

Compound 1 (87 mg, ‘73 μmol) was combined with MeCN (1 mL) and 4N HCl indioxane (0.3 mL) and stirred for 10 minutes, then concentrated underreduced pressure to yield Compound (2).

Compound 2 (33.7 mg, 83 μmol), HATU (38.0 mg, 100 μmol),3-Hydroxy-isoxazole-5-carboxylic acid (12.3 mg, 108 μmol) and DMF (0.5mL) were combined, followed by DIPEA (43.7 μL, 250 μmol). The resultingmixture was stirred for 2 hours. EtOAc was added, then saturated aqueousNH₄Cl. The mixture was then concentrated under reduced pressure. Theresidue was combined with THF (0.6 mL) and NaOH (326 μL, 326 μmol) witha few drop of MeOH, and stirred at 60° C. for 2 hours. The mixture wasthen concentrated under reduced pressure. The residue was dissolved inAcOH and purified by preparative HPLC to yield the title compound (8mg). MS m/z [M+H]⁺ calc'd for C₂₅H₂₇ClN₂O₆, 487.16; found 486.9.

6N:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-ethoxymethyl-4-[(3-hydroxyisoxazole-5-carbonyl)amino]-2-methylpentanoicAcid

(2S,4R)-4-t-Butoxycarbonylamino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-ethoxymethyl-2-methylpentanoicacid benzyl ester (720 mg, 1.2 mmol) was combined with MeCN (6 mL),followed by the addition of 4N HCl in dioxane (5 mL). The resultingmixture was stirred for 10 minutes then concentrated under reducedpressure to yield Compound 1.

3-Hydroxyisoxazole-5-carboxylic acid (53.3 mg, 413 μmol) was combinedwith HATU (157 mg, 413 μmol) and DMF (0.5 mL mL) and the resultingmixture was stirred for 20 minutes. N-ethyl-N-isopropylpropan-2-amine (1eq.) was added and the resulting mixture was stirred for 1 minute.Compound 1 (100 mg, 207 μmol) pre-dissolved in DMF (2 mL) and DIPEA (108μl, 620 μmol) was then added and the resulting mixture was stirredovernight then concentrated under reduced pressure. The material wasthen purified by normal phase (40% EtOAc/hexanes) to yield Compound 2(90 mg).

Compound 2 (90 mg, 151 μmol) was combined with palladium on carbon (16.1mg, 30 μmol) dissolved in EtOAc (1 mL) and AcOH (1 mL). The resultingsolution was degassed in vacuo and purged with hydrogen gas. Thesolution was stirred for 2 hours. The hydrogen gas was removed and thesolution was purged with nitrogen. The solution was filtered, the excesssolvent was removed from the filtrate and the residue was purified byreverse phase chromatography to yield the title compound (60 mg). MS m/z[M+H]⁺ calc'd for C₂₅H₂₆ClFN₂O₆, 505.15; found 505.

6O:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-ethoxymethyl-4-[(3-ethylisoxazole-5-carbonyl)amino]-2-methylpentanoicAcid

(2S,4R)-4-t-Butoxycarbonylamino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-ethoxymethyl-2-methylpentanoicacid (220 mg, 445 μmol) was combined with MeCN (5 mL), followed by theaddition of 4N HCl in dioxane (4 mL). The resulting mixture was stirredfor 10 minutes then concentrated under reduced pressure to yieldCompound 1.

3-Ethylisoxazole-5-carboxylic acid (6.0 mg, 42 μmol) was combined withHATU (16.1 mg, 42 μmol) and DMF (0.5 mL) and the resulting mixture wasstirred for 10 minutes. N-ethyl-N-isopropylpropan-2-amine (1 eq.) wasadded and the resulting mixture was stirred for 1 minute. Compound 1 (20mg, 51 μmol) pre-dissolved in DMF (0.5 mL) and DIPEA (22.2 μL, 127 μmol)was then added the resulting mixture was stirred for 30 minutes. Themixture was then concentrated under reduced pressure, removing abouthalf of the solvent. AcOH was added to the residue, and the material waspurified by preparative HPLC to yield the title compound (2.5 mg). MSm/z [M+H]⁺ calc'd for C₂₇H₃₀ClFN₂O₅, 517.18; found 518.2.

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, thefollowing compounds can also be prepared.

6P:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-4-[(3-hydroxyisoxazole-5-carbonyl)amino]-2-hydroxymethyl-2-methylpentanoicAcid

6Q:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-4-[(3-ethylisoxazole-5-carbonyl)amino]-2-methoxymethyl-2-methylpentanoicAcid

Example 7 7A:(2S,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-[(5-ethoxy-1H-pyrazole-3-carbonyl)amino]2-hydroxymethyl-2-methylpentanoicAcid

5-Ethoxy-1H-pyrazole-3-carboxylic acid (10 mg, 32 μmol) was combinedwith HATU (12 mg, 32 μmol) in DMF (0.2 mL) and the resulting mixture wasstirred for 5 minutes. DIPEA (17 μL, 96 μmol) and(2S,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (79 mg, 38 μmol) pre-dissolved in DMF were added and the resultingmixture was stirred for 15 minutes then concentrated. The residue wasdissolved in AcOH and purified by preparative HPLC to yield the titlecompound as a TFA salt (1 mg). MS m/z [M+H]⁺ calc'd for C₂₅H₂₈ClN₃O₅,486.17; found 486.2.

7B:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-4-[(5-ethoxy-1H-pyrazole-3-carbonyl)-amino]-2-hydroxymethyl-2-methylpentanoicAcid

5-Ethoxy-1H-pyrazole-3-carboxylic acid (8.5 mg, 55 μmol) was combinedwith HATU (21 mg, 55 μmol) in DMF (0.3 mL) and the resulting mixture wasstirred for 5 minutes. DIPEA (29 μL, 164 μmol) and(2S,4R)-4-amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (20 mg, 55 μmol) pre-dissolved in DMF (0.5 mL) were added and theresulting mixture was stirred for 10 minutes then concentrated. Theresidue was dissolved in AcOH and purified by preparative HPLC to yieldthe title compound as a TFA salt (2 mg). MS m/z [M+H]⁺ calc'd forC₂₅H₂₇ClFN₃O₅, 504.16; found 503.9.

7C:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-4-[(5-isopropyl-2H-pyrazole-3-carbonyl)amino]-2-methylpentanoicAcid

5-Isopropyl-2H-pyrazole-3-carboxylic acid (8 mg, 55 μmol) and HATU (20.8mg, 55 μmol) were combined in DMF (0.2 mL) and allowed to stand at roomtemperature for 10 minutes.(2S,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (20 mg, 55 μmol) in DMF and DIPEA (28.6 μL, 164 μmol) were added,and the resulting mixture was stirred for 20 minutes at roomtemperature. The mixture was concentrated under reduced pressure and theresidue was dissolved in AcOH purified by preparative HPLC to yield thetitle compound as a TFA salt (2 mg). MS m/z [M+H]⁺ calc'd forC₂₆H₂₉ClFN₃O₄, 502.18; found 503.2.

7D:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-4-[(5-ethoxy-2H-pyrazole-3-carbonyl)-amino]-2-methoxymethyl-2-methylpentanoicAcid

(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-4-[(5-ethoxy-2H-pyrazole-3-carbonyl)amino]-2-methoxymethyl-2-methylpentanoicacid ethyl ester (11 mg) in THF (1 mL) was combined with 1N NaOH (0.3mL) and the resulting mixture was stirred at 60° C. for 3 hours. AcOHwas added and the product was purified (reverse phase) to yield thetitle compound as a TFA salt (4 mg). MS m/z [M+H]⁺ calc'd forC₂₆H₂₉ClFN₃O₅, 518.18; found 518.

7E:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-4-[(5-methoxy-1H-pyrazole-3-carbonyl)amino]-2-methylpentanoicAcid

(2S,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid ethyl ester (30 mg, 76 μmol), HATU (29.0 mg, 0.076 mmol), and DIPEA(39.9 μl, 0.228 mmol), were combined with1h-[1,2,4]triazole-3-carboxylic acid (8.61 mg, 0.076 mmol) in DMF (0.5mL). The resulting mixture was stirred for 2 hours then concentratedunder reduced pressure. The residue was combined with THF (1 mL) andNaOH (456 μL, 456 μmol) and stirred for 2 days at 40° C. The reactionwas quenched with AcOH and the material was purified by preparative HPLCto yield the title compound as a TFA salt (16.4 mg). MS m/z [M+H]⁺calc'd for C₂₄H₂₅ClFN₃O₅, 490.15; found 490.2.

7F:(2S,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-4-[(5-isobutyl-2H-pyrazole-3-carbonyl)amino]-2-methylpentanoicAcid

5-Isobutyl-2H-pyrazole-3-carboxylic acid (10.1 mg, 60 μmol) and HATU(22.9 mg, 60 μmol) were combined then stirred in DMF (1 mL) for 15minutes at room temperature.(2S,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (20 mg, 55 μmol) and Et₃N (38 μL, 273 μmol) were premixed togetherand then added to the reaction solution. The resulting mixture wasstirred for 1 hour at room temperature. The solvent was removed in vacuoand the residue was purified by preparative HPLC to yield the titlecompound as a TFA salt (13.3 mg). MS m/z [M+H]⁺ calc'd forC₂₇H₃₁ClFN₃O₄, 516.20; found 516.2.

Example 8 8A:(2S,4R)-4-[(5-Acetyl-2H-pyrazole-3-carbonyl)amino]-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicAcid

5-Acetyl-2H-pyrazole-3-carboxylic acid (8 mg, 55 μmol) and HATU (20.8mg, 55 μmol) were combined in DMF (0.2 mL) and allowed to stand at roomtemperature for 10 minutes.(2S,4R)-4-Amino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (20 mg, 55 μmol) in DMF and DIPEA (28.6 μL, 164 μmol) were added,and the resulting mixture was stirred for 20 minutes at roomtemperature. The mixture was concentrated under reduced pressure and theresidue was dissolved in AcOH purified by preparative HPLC to yield thetitle compound as a TFA salt (2.1 mg). MS m/z [M+H]⁺ calc'd forC₂₅H₂₅ClFN₃O₅, 502.15; found 503.2.

8B:(2S,4R)-4-[(5-Acetyl-2H-pyrazole-3-carbonyl)amino]-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-methoxymethyl-2-methylpentanoicAcid

Into a vial was added(2S,4R)-4-t-butoxycarbonylamino-5-(5′-chloro-2′-fluoro-biphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid ethyl ester (415 mg, 840 μmol), tetrabutylammonium hydrogen sulfate(57 mg, 168 μmol), DCM (1 mL) and NaOH (588 μL, 5.9 mmol), followed bydiethylsulfate (518 mg, 3.4 mmol). The reaction vessel was capped andstirred vigorously overnight. The mixture was extracted with DCM andwater, purified (normal phase chromatography 0-60% EtOAc:hexanes), thenconcentrated under reduced pressure to yield Compound 1 (220 mg).

Compound 1 (88 mg, 173 μmol) in MeCN (1 mL) was combined with 4N HCl indioxane (0.3 mL). The mixture was stirred for 10 minutes thenconcentrated under reduced pressure to yield Compound 2.

Compound 2 (10 μg, 0.03 μmol) in DMF (0.5 mL) was combined with HATU (11μg, 0.03 μmol) and 5-acetyl-2H-pyrazole-3-carboxylic acid (4 μg, 0.03μmol), and the resulting mixture was stirred for 5 minutes. DIPEA (0.01μl, 0.07 μmol) was added and the mixture was stirred for 20 minutes. Thesolvent was evaporated to yield Compound 3, which was used withoutfurther purification.

Compound 3 (11 mg, 20 μmol) was combined with THF (1 mL) and 1N NaOH(0.3 mL). The resulting mixture was stirred at 60° C. for 3 hours. AcOHwas added and the product was purified by reverse phase HPLC to yieldthe title compound as a TFA salt (2 mg). MS m/z [M+H]⁺ calc'd forC₂₆H₂₇ClFN₃O₅, 516.16; found 516.

8C:(2S,4R)-4-[(5-Acetyl-1H-pyrazole-3-carbonyl)amino]-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-ethoxymethyl-2-methylpentanoicAcid

(2S,4R)-4-t-Butoxycarbonylamino-5-(5′-chloro-2′-fluorobiphenyl-4-yl)-2-ethoxymethyl-2-methylpentanoicacid (220 mg, 445 μmol) was combined with MeCN (5 mL), followed by theaddition of 4N HCl in dioxane (4 mL). The resulting mixture was stirredfor 10 minutes then concentrated under reduced pressure to yieldCompound 1.

5-Acetyl-1H-pyrazole-3-carboxylic acid (6.5 mg, 42 μmol) was combinedwith HATU (16.1 mg, 42 μmol) and DMF (0.5 mL) and the resulting mixturewas stirred for 10 minutes. N-ethyl-N-isopropylpropan-2-amine (1 eq.)was added and the resulting mixture was stirred for 1 minute. Compound 1(20 mg, 51 μmol) pre-dissolved in DMF (0.5 mL) and DIPEA (22.2 μL, 127μmol) was then added the resulting mixture was stirred for 30 minutes.The mixture was then concentrated under reduced pressure, removing abouthalf of the solvent. AcOH was added to the residue, and the material waspurified by preparative HPLC to yield the title compound as a TFA salt(3.1 mg). MS m/z [M+H]⁺ calc'd for C₂₇H₂₉ClFN₃O₅, 530.18; found 531.2.

Assay In Vitro Assays for the Quantitation of Inhibitor Potencies (IC₅₀)at Human and Rat NEP, and Human ACE

The inhibitory activities of compounds at human and rat neprilysin (EC3.4.24.11; NEP) and human angiotensin converting enzyme (ACE) weredetermined using in vitro assays as described below.

Extraction of NEP Activity from Rat Kidneys

Rat NEP was prepared from the kidneys of adult Sprague Dawley rats.Whole kidneys were washed in cold phosphate buffered saline (PBS) andbrought up in ice-cold lysis buffer (1% Triton X-114, 150 mM NaCl, 50 mMtris(hydroxymethyl) aminomethane (Tris) pH 7.5; Bordier (1981) J. Biol.Chem. 256: 1604-1607) in a ratio of 5 mL of buffer for every gram ofkidney. Samples were homogenized on ice using a polytron hand heldtissue grinder. Homogenates were centrifuged at 1000×g in a swingingbucket rotor for 5 minutes at 3° C. The pellet was resuspended in 20 mLof ice cold lysis buffer and incubated on ice for 30 minutes. Samples(15-20 mL) were then layered onto 25 mL of ice-cold cushion buffer (6%w/v sucrose, 50 mM pH 7.5 Tris, 150 mM NaCl, 0.06%, Triton X-114),heated to 37° C. for 3-5 minutes and centrifuged at 1000×g in a swingingbucket rotor at room temperature for 3 minutes. The two upper layerswere aspirated off, leaving a viscous oily precipitate containing theenriched membrane fraction. Glycerol was added to a concentration of 50%and samples were stored at −20° C. Protein concentrations werequantitated using a BCA detection system with bovine serum albumin (BSA)as a standard.

Enzyme Inhibition Assays

Recombinant human NEP and recombinant human ACE were obtainedcommercially (R&D Systems, Minneapolis, Minn., catalog numbers 1182-ZNand 929-ZN, respectively). The fluorogenic peptide substrateMca-D-Arg-Arg-Leu-Dap-(Dnp)-OH (Medeiros et al. (1997) Braz. J Med.Biol. Res. 30:1157-62; Anaspec, San Jose, Calif.) andAbz-Phe-Arg-Lys(Dnp)-Pro-OH (Araujo et al. (2000) Biochemistry39:8519-8525; Bachem, Torrance, Calif.) were used in the NEP and ACEassays respectively.

The assays were performed in 384-well white opaque plates at 37° C.using the fluorogenic peptide substrates at a concentration of 10 μM inAssay Buffer (NEP: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% polyethyleneglycol sorbitan monolaurate (Tween-20), 10 μM ZnSO₄; ACE: 50 mM HEPES,pH 7.5, 100 mM NaCl, 0.01% Tween-20, 1 μM ZnSO₄). The respective enzymeswere used at concentrations that resulted in quantitative proteolysis of1 μM of substrate after 20 minutes at 37° C.

Test compounds were assayed over the range of concentrations from 10 μMto 20 pM. Test compounds were added to the enzymes and incubated for 30minute at 37° C. prior to initiating the reaction by the addition ofsubstrate. Reactions were terminated after 20 minutes of incubation at37° C. by the addition of glacial acetic acid to a final concentrationof 3.6% (v/v).

Plates were read on a fluorometer with excitation and emissionwavelengths set to 320 nm and 405 nm, respectively. Inhibition constantswere obtained by nonlinear regression of the data using the equation(GraphPad Software, Inc., San Diego, Calif.):ν=ν₀/[1+(I/K′)]where ν is the reaction rate, ν₀ is the uninhibited reaction rate, I isthe inhibitor concentration and K′ is the apparent inhibition constant.

The compound of formula I′ where R^(a) and R^(b) are H was tested inthis assay and found to have a pK_(i) value at human NEP of ≥9.0. Thefollowing compounds were found to have pK_(i) values at human NEP asfollows:

Ex. pK_(i) 1A n.d. 1B n.d. 1C n.d. 1D ≥9.0 1E ≥9.0 1F ≥9.0 1G n.d. 1Hn.d. 1I 8.5-9.0 1J 8.5-9.0 2A 8.5-9.0 2B ≥9.0

Ex. pK_(i) 2C ≥9.0 2D ≥9.0 2E 8.5-9.0 2F ≥9.0 2G ≥9.0 2H 8.0-8.5 2I n.d.2J 8.5-9.0 2K ≥9.0 2L ≥9.0 2M ≥9.0 2N ≥9.0 2O ≥9.0 2P ≥9.0 2Q 8.5-9.0 2R≥9.0 2S 8.0-8.5The remaining compounds were not tested (n.d.) since activity would notbe expected in this in vitro assay; however, based upon the activity ofthe active forms, the corresponding prodrugs are expected to have invivo NEP activity.

The compound of formula I′ where R^(a) is H and R^(b) is F (Example 3A)and the compound of formula I′ where R^(a) is F and R^(b) is H (Example3B) were both tested in this assay and found to have a pK_(i) value athuman NEP of ≥9.0. Based upon the activity of these active forms, thecorresponding prodrug compounds are expected to have in vivo NEPactivity.

The compound of formula II where R^(a) is F, R^(b) is H, R² is H, and R⁷is H (Example 4A) was tested in this assay and found to have a pK_(i)value at human NEP of ≥9.0. Based upon the activity of this active form,the corresponding prodrug compounds are expected to have in vivo NEPactivity. The following compounds were also found to have pK_(i) valuesat human NEP:

Ex. pK_(i) 4B ≥9.0 4C ≥9.0

The compound of formula IIIa, where R^(a) is F, R^(b) is H, R² is H, andR⁷ is H (Example 5A), and the compounds of formula IIIb, where R^(a) isF, R^(b) is H, R² is H, and R⁷ is H (Example 5B) were both tested inthis assay and found to have a pK_(i) value at human NEP of ≥9.0. Basedupon the activity of these active forms, the corresponding prodrugcompounds are expected to have in vivo NEP activity. In addition, thefollowing compounds were also found to have pK_(i) values at human NEP:

Ex. pK_(i) 5C ≥9.0 5D ≥9.0

The compound of formula V, where R^(a) is H, R^(b) is Cl, R² is H, R³ is—OCH₃, and R⁷ is H (Example 6A) and the compound of formula V′ (whereR^(a) and R^(b) are H and R³ is —OH; Example 6D) were both tested inthis assay and found to have a pK_(i) value at human NEP of ≥9.0. Basedupon the activity of these active forms, the corresponding prodrugcompounds are expected to have in vivo NEP activity. In addition, thefollowing compounds were also found to have pK_(i) values at human NEP:

Ex. pK_(i) 6B 7.0-8.0 6C n.d. 6E n.d. 6F n.d. 6G ≥9.0 6H ≥9.0 6I ≥9.0 6J≥9.0 6K 8.5-9.0 6L ≥9.0 6M ≥9.0 6N ≥9.0 6O ≥9.0The remaining compounds were either not tested or did not show activityin this in vitro assay (n.d.) since activity would not be expected;however, based upon the activity of the active forms, thesecorresponding prodrugs are expected to have in vivo NEP activity.

Compounds of formula VI were tested in this assay and found to havepK_(i) values at human NEP as follows:

Ex. pK_(i) 7A ≥9.0 7B ≥9.0 7C ≥9.0 7D 8.5-9.0 7E ≥9.0 7F ≥9.0Based upon the activity of these active forms, the corresponding prodrugcompounds are expected to have in vivo NEP activity.

Compounds of formula VII were tested in this assay and found to havepK_(i) values at human NEP as follows:

Ex. pK_(i) 8A ≥9.0 8B 8.5-9.0 8C ≥9.0Based upon the activity of this active form, the corresponding prodrugcompounds are expected to have in vivo NEP activity.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

What is claimed is:
 1. A method for treating hypertension, the methodcomprising administering to patient a therapeutically effective amountof a compound of the following structure:

or a pharmaceutically acceptable salt thereof; wherein the hypertensionis selected from the group consisting of primary hypertension, secondaryhypertension, hypertension with accompanying renal disease, severehypertension with or without accompanying renal disease, pulmonaryhypertension, and resistant hypertension.
 2. The method according toclaim 1, wherein the hypertension is resistant hypertension.
 3. A methodfor treating portal hypertension, the method comprising administering topatient a therapeutically effective amount of a compound of thefollowing structure:

or a pharmaceutically acceptable salt thereof.